Microneedle comprising silk fibroin applied to a dissolvable base

ABSTRACT

Microneedle and microneedle devices including implantable silk-based tips for sustained dermal delivery of a vaccine, kits, as well as methods of manufacturing and using the same are described herein. In other embodiments, compositions and methods for controlled- or sustained-administration of a vaccine (e.g., an influenza vaccine) to provide improved immunogenicity and/or broad-spectrum immunity to a subject are also described.

RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 62/652,275 filed Apr.4, 2018, the contents of which is incorporated herein by reference inits entirety.

GOVERNMENT SUPPORT

This invention was made with support from the federal government underGrant No. 1632434 awarded by the National Science Foundation as a SBIRPhase II award. The U.S. government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to compositions and devices forachieving a controlled- or sustained-release of a vaccine in a subject,and methods of making and using the same.

BACKGROUND OF THE INVENTION

Recent studies have shown that modulating the kinetics of antigenpresentation to mimic those of a natural infection can drive more potentimmune responses. The use of microneedles has been investigated in thedelivery of therapeutic agents, including vaccines. Traditionalmaterials used in the fabrication of microneedles, including silicon,metals, dextrin, glass, ceramic, maltose, galactose, and syntheticpolymers, are known to be associated with various limitations thatcompromise their production and limit their performance (see, e.g.,Donnelly et al. Drug Deliv. 17(4): 187-207, 2010).

The use of silk and silk-based materials in the fabrication ofmicroneedles for controlled and sustained vaccine delivery has beenexplored to a lesser extent. However, silk fibroin has suitableproperties for use in microneedle fabrication, including all-aqueousprocessing, mechanical strength, biocompatibility, and the ability tostabilize various macromolecules of biological origin. There remains astrong need for effective vaccine-delivery compositions, devices (e.g.,silk-based microneedles), and methods capable of controlling and/orsustaining vaccine release to enhance an immune response (e.g., acellular immune response and/or a humoral immune response) in a subject,and improved approaches to the manufacture of such compositions anddevices (e.g., silk-based microneedles).

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery thatmodulating the kinetics of antigen presentation via, e.g., controlled-and/or sustained release compositions and devices (e.g., microneedles,e.g., silk-based microneedles, and microneedles devices) comprising avaccine as described herein, e.g., a viral vaccine such as an influenzavaccine, can drive a more potent and/or lasting immune response (e.g., amore potent and/or lasting cellular immune response and/or humoralimmune response) in a subject, e.g., as compared to the administrationof single-dose or bolus administration of the vaccine. In someembodiments, controlled- or sustained-release of a vaccine as describedherein can be used to achieve broad spectrum immunity in a subject.

In some embodiments, the microneedles and microneedles devices describedherein demonstrate controlled- or sustained-release of a vaccine (e.g.,an influenza vaccine) for at least about 1-2 weeks (e.g., for at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days), whichresults in one or more of improved immunogenicity, an enhanced immuneresponse, and/or broad-spectrum immunity.

In other embodiments, methods, formulations, compositions, articles,devices, and/or preparations for administering a vaccine (e.g., aninfluenza vaccine) that provide improved immunogenicity, an enhancedimmune response, and/or a broad-spectrum immunity to a subject are alsodisclosed. Accordingly, disclosed herein are compositions, preparations,devices (e.g., microneedles and microneedles devices), kits forcontrolled- and/or sustained release of a vaccine, in a subject, as wellas methods of making and using the same.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following embodiments (E).

E1. A microneedle comprising:

(i) a backing,

(ii) a dissolvable base comprising a component other than poly acrylicacid (PAA), e.g., other than a solution of about 35% PAA. Inembodiments, the dissolvable base comprises a component, e.g., one ormore water-soluble components, having improved biocompatibility e.g., ina subject, compared to PAA. In some embodiments, the dissolvable basecomprises a component, e.g., a water soluble components, that has a pHsimilar to that of a biological barrier into which it will be dissolved,e.g., has a pH of about 4.0-8. In embodiments, the dissolvable basecomprises one, two, three, four, five, six, seven, eight, or more (e.g.,all) of gelatin, polyethylene glycol (PEG), sucrose,carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA), hyaluronate, maltose, and methyl cellulose. In someembodiments, the dissolvable base does not comprise a therapeutic agent,as described herein. In some embodiments, the dissolvable base isapplied to the backing,

(iii) a microneedle tip, e.g., an implantable sustained-release tip,comprising a therapeutic agent and a silk fibroin. In some embodiments,the tip is applied to the dissolvable base,

wherein the microneedle is configured to implant the tip into abiological barrier, e.g., the skin of a subject, e.g., a human subject,e.g., at a depth (e.g., a max penetration depth of the distal part oftip) of between about 100 μm and about 600 μm,

wherein the tip comprises a silk fibroin, e.g., a regenerated silkfibroin and/or a recombinant silk fibroin.

In embodiments, the therapeutic agent in the tip is chosen from anantigen, an immunogen or a vaccine (e.g., an influenza vaccine). Inembodiments, the therapeutic agent is present in an amount sufficient toinduce an immune response, e.g., a humoral and/or cellular immuneresponse.

E2. A microneedle comprising:

(i) a backing,

(ii) a dissolvable base comprising one, two, three, four, five, six,seven, eight, or more (e.g., all) of gelatin, polyethylene glycol (PEG),sucrose, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP),polyvinyl alcohol (PVA), hyaluronate, maltose, and methyl celluloseapplied to the backing,

(iii) a microneedle tip, e.g., an implantable sustained-release tip,comprising a silk fibroin applied to the dissolvable base,

wherein the microneedle is configured to implant the tip into the skinof a subject, e.g., a human subject, at a depth (e.g., a max penetrationdepth of the distal part of tip) of between about 100 μm and about 600μm,

wherein the tip comprises a silk fibroin, e.g., a regenerated silkfibroin and/or a recombinant silk fibroin,

wherein the tip further comprises a therapeutic agent, e.g., an antigen,an immunogen or a vaccine (e.g., an influenza vaccine), e.g., in anamount sufficient to induce an immune response, e.g., a humoral and/orcellular immune response.

E3. The microneedle of embodiment E1 or E2, wherein the dissolvable basecomprises one of gelatin, polyethylene glycol (PEG), sucrose,carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA), hyaluronate, maltose, and methyl cellulose.E4. The microneedle of embodiment E1 or E2, wherein the dissolvable baseis comprised of two of gelatin, PEG, sucrose, CMC, PVP, PVA,hyaluronate, maltose, and methyl cellulose.E5. The microneedle of embodiment E1 or E2, wherein the dissolvable basecomprising three of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate,maltose, and methyl cellulose.E6. The microneedle of embodiment E1 or E2, wherein the dissolvable basecomprising four of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate,maltose, and methyl cellulose.E7. The microneedle of embodiment E1 or E2, wherein the dissolvable basecomprising five of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate,maltose, and methyl cellulose.E8. The microneedle of embodiment E1 or E2, wherein the dissolvable basecomprising six of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate,maltose, and methyl cellulose.E9. The microneedle of embodiment E1 or E2, wherein the dissolvable basecomprising seven of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate,maltose, and methyl cellulose.E10. The microneedle of embodiment E1 or E2, wherein the dissolvablebase comprising gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate,maltose, and methyl cellulose.E11. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprising gelatin and sucrose.E12. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises CMC.E13. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises PVP.E14. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises PVA.E15. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises about PVP and PVA.E16. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises PVP, PVA, and sucrose.E17. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base does not comprise poly(acrylic acid) (PAA).E18. The microneedle of any one of the preceding embodiments, whereinthe implantable sustained-release tip is configured to release a vaccineinto the skin of the subject over a period of time comprising at leastabout 4 days (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 or more days, e.g., between about 4 days and about 14 days, e.g.,between about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks).E19. The microneedle of any one of the preceding embodiments, whereinthe implantable sustained-release tip is configured to release a vaccineinto the skin of the subject over a period of time comprising betweenabout 1 week to about 2 weeks (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, or 14 days).E20. The microneedle of any one of the preceding embodiments, whereinimmune response comprises a cellular and/or humoral immune responsecomprising: (i) an elevated hemagglutination inhibition (HAI) antibodytiter detectable in the blood of the subject, e.g., detectable at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, and/or 52-weeks or morepost immunization, optionally wherein the elevated HAI antibody titer isto a drifted influenza A, B, C, and/or D strain;

(ii) an elevated anti-influenza IgG titer detectable in the blood of thesubject, e.g., detectable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,and/or 12-months or more post immunization, optionally wherein theelevated anti-influenza IgG titer is to a drifted influenza A, B, C,and/or D strain; and/or

(iii) a level of antibody secreting plasma cells (ASC) against thevirus, e.g., the influenza virus, e.g., the drifted influenza A, B, C,and/or D strain, detectable in the bone marrow of the subject, e.g.,detectable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51,and/or 52-weeks or more post immunization.

E21. The microneedle of any one of the preceding embodiments, wherein:

(i) an elevated hemagglutination inhibition (HAI) antibody titer isdetectable in the blood of the subject for the duration of a completeflu season post immunization, optionally wherein the elevated HAIantibody titer is to a drifted influenza A, B, C, and/or D strain;and/or

(ii) the percent seroconversion, e.g., based on the elevated HAIantibody titer detectable in the blood of the subject, e.g., at 6-monthpost immunization is greater than about 20% (e.g., 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more,e.g., 100%).

E22. The microneedle of any one of the preceding embodiments, wherein:

(i) the immune response is a cellular immune response comprising anincrease in the number of IFNγ secreting cells in the blood of thesubject, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51,and/or 52-weeks or more post immunization; and/or

(ii) the elevated HAI antibody titer, the elevated anti-influenza IgGtiter, the level of antibody secreting plasma cells (ASC) against thevirus, and/or the level of IFNγ secreting cells detectable in thesubject is greater (e.g., 1-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold,14-fold, or 15-fold or more greater) as compared to the administrationof a single-dose or a bolus administration of the vaccine.

E23. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises between about 10% and about 70% gelatin(e.g., hydrolyzed gelatin) (e.g., about 10%, about 20%, about 30%, about40%, about 50%, about 60%, or about 70% gelatin).E24. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises between about 1% and about 35% sucrose(e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%,about 30%, or about 35% sucrose).E25. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises between about 1% and about 35% CMC (e.g.,about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about30%, or about 35% CMC).E26. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises between about 10% and about 70% PVP(e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,or about 70% PVP).E27. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises between about 1% and about 35% PVA (e.g.,e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%,about 30%, or about 35% PVA).E28. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises about 40% hydrolyzed gelatin and about10% sucrose w/v.E29. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises up to about 50% w/v of PVP (e.g., PVP of10 kD MW).E30. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises up to about 20% PVA (e.g., 87% hydrolyzedPVA at 13 kD MW).E31. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises CMC at up to about 10%.E32. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises about 1% CMC (e.g., low-viscosity CMC).E33. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises about 30% PVP and about 10% PVA.E34. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises about 37% PVP, about 5% PVA, and about15% sucrose.E35. The microneedle of any one of the preceding embodiments, whereinthe implantable sustained-release tip comprises silk fibroin at about 1%w/v to about 10% w/v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w/v,or a silk fibroin having a molecular weight distribution according toFIG. 5, or, comprises silk fibroin in an amounta between about 20 μg toabout 245 μg, e.g., per 121 microneedle array).E36. The microneedle of any one of the preceding embodiments, whereinthe implantable sustained-release tip comprises a vaccine formulated ina 1% w/v to about 10% w/v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%w/v) of 10 MB silk fibroin solution, or a silk fibroin solutionaccording to FIG. 5.E37. The microneedle of any one of the preceding embodiments, whereinthe implantable sustained-release tip comprises a vaccine formulated ina 1% w/v to about 10% w/v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%w/v) of 60 MB silk fibroin solution, or a silk fibroin solutionaccording to FIG. 5, e.g., a 100 kDa to 200 kDa (e.g., about 153 kDa)silk fibroin solution.E38. The microneedle of any one of the preceding embodiments, whereinthe implantable sustained-release tip comprises a vaccine formulated ina 1% w/v to about 10% w/v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%w/v) of 120 MB silk fibroin solution, or a silk fibroin solutionaccording to FIG. 5, e.g., a 70 kDa to 150 kDa (e.g., about 100 kDa)silk fibroin solution.E39. The microneedle of any one of the preceding embodiments, whereinthe implantable sustained-release tip comprises a vaccine formulated ina 1% w/v to about 10% w/v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%w/v) of 180 MB silk fibroin solution, or a silk fibroin solutionaccording to FIG. 5, e.g., a 36 kDa to 100 kDa (e.g., about 71 kDa) silkfibroin solution.E40. The microneedle of any one of the preceding embodiments, whereinthe implantable sustained-release tip comprises a vaccine formulated ina 1% w/v to about 10% w/v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%w/v) of 480 MB silk fibroin solution, or a silk fibroin solutionaccording to FIG. 5, e.g., a 1 kDa to 60 kDa (e.g., about 16 kDa) silkfibroin solution.E41. The microneedle of embodiment E37, wherein the implantablesustained-release tip comprises a 5% wt/vol of 60 MB silk fibroinsolution.E42. The microneedle of any one of the preceding embodiments, whereinthe implantable sustained-release tip comprises a standard human dose ofa vaccine.E43. The microneedle of any one of the preceding embodiments, whereinthe standard dose of the vaccine (e.g., influenza vaccine) comprisesbetween about 0.1 μg and about 65 μg per strain, e.g., 0.2 μg and about50 μg per strain (e.g., about each of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 μg per strain.E44. The microneedle of embodiment E42 or E43, wherein the implantablesustained-release tip comprises at least about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, or 25% or more of the standard dose.E45. The microneedle of any one of embodiment E42-E44, wherein theimplantable sustained-release tip comprises about 0.1 μg to about 65 μgof vaccine (e.g., about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9μg, about 1 μg, about 1 μg to about 10 μg, about 10 μg to about 20 μg,about 20 μg to about 30 μg, about 30 μg to about 40 μg, about 40 μg toabout 50 μg, about 50 μg to about 65 μg of a vaccine).E46. The microneedle of any one of the preceding embodiments, whereinthe length of the microneedle is between about 350 μm to about 1500 μm((e.g., about 350 μm, about 400 μm, about 450 μm, about 500 μm, about550 μm, about 600 μm, about 650 μm, about 700 μm, about 750 μm, about800 μm, about 850 μm, about 900 μm, about 950 μm, about 1000 μm, about1050 μm, about 1100 μm, about 1150 μm, about 1200 μm, about 1250 μm,about 1300 μm, about 1350 μm, about 1400 μm, about 1450 μm, about 1500μm).E47. The microneedle of any one of the preceding embodiments, whereinthe height of the implantable sustained-release tip may extend toapproximately half of the full height of the microneedle.E48. The microneedle of any one of the preceding embodiments, whereinthe height of the implantable sustained-release tip is between about 75μm to about 475 μm (e.g., about 75, about 100 μm, about 125 μm, about150 μm, about 175 μm, about 200 μm, about 225 μm, about 250 μm, about275 μm, about 300 μm, about 325 μm, about 375 μm, about 400 μm, about425 μm, or about 475 μm).E49. The microneedle of any one of the preceding embodiments, whereinthe implantable sustained-release tip comprises a tip radius betweenabout 0.5 μm to about 25 μm (e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 μm).E50. The microneedle of any one of the preceding embodiments, whereinthe implantable sustained-release tip comprises a tip radius betweenabout 5 μm to about 10 μm (e.g., about 5, 6, 7, 8, 9, or 10 μm).E51. The microneedle of any one of the preceding embodiments, whereinthe implantable sustained-release tip comprises an angle between about 5degrees and about 45 degrees (e.g., about 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, or 45 degrees).E52. The microneedle of any one of the preceding embodiments, whereinthe backing is chosen from a solid support, e.g., a paper-basedmaterial, a plastic material, a polymeric material, or a polyester-basedmaterial (e.g., a Whatman 903 paper, a polymeric tape, a plastic tape,an adhesive-backed polyester tape, or other medical tape).E53. The microneedle of any one of the preceding embodiments, whereinthe implantable sustained-release tip comprises an influenza vaccine,e.g., a univalent (e.g., monovalent) or multivalent influenza vaccine(e.g., a tetravalent or quadrivalent influenza vaccine).E54. The microneedle of embodiment E53, wherein the influenza vaccinecomprises an influenza A vaccine, an influenza B vaccine, an influenza Cvaccine, and/or an influenza D vaccine.E55. The microneedle of embodiment E53 or E54, wherein the influenzavaccine comprises an influenza A vaccine, optionally wherein theinfluenza A vaccine is a H1N1 (e.g., A/Michigan and/or A/California)vaccine and/or a H3N2 (e.g., A/Hong Kong and/or A/Switzerland) vaccine.E56. The microneedle of any one of embodiments E53-E55, wherein theinfluenza vaccine comprises an influenza B vaccine, optionally whereinthe influenza B vaccine is an B/Yamagata lineage (e.g., B/Phuket) and/orthe B/Victoria lineage (e.g., B/Brisbane) vaccine.E57. The microneedle of any one of embodiments E53-E56, wherein theinfluenza vaccine comprises an influenza A vaccine (e.g., a H1N1 vaccineand/or a H3N2 vaccine) and an influenza B vaccine (e.g., an B/Yamagatalineage and/or the B/Victoria lineage vaccine).E58. A device, e.g., an array or patch, comprising a plurality ofmicroneedles (e.g., two or more microneedles as described herein), e.g.,a plurality of microneedles according to any one of embodiments E1-E57.E59. The device of embodiment E58, wherein the microneedles of theplurality are the same, e.g., comprise the same implantablesustained-release tip, e.g., comprising the same therapeutic agent,e.g., the same immunogen, antigen or vaccine.E60. The device of embodiment E58, wherein two or more of themicroneedles of the plurality are different, e.g., comprise two or moredifferent implantable sustained-release tips, e.g., comprising two ormore therapeutic agents, e.g., comprising a combination of two or moreimmunogens, antigens or vaccines, with or without one or more adjuvants.E61. The device of embodiment E60, which comprises at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or more of a first implantablesustained-release tip relative to a further (e.g., second, third,fourth, fifth) implantable sustained-release tip.E62. The device of embodiment E60, wherein a total dosage amount (e.g.,a standard dose) of a vaccine, antigen, and/or immunogen is dividedbetween the plurality of microneedles (e.g., within a patch), such thatthe implantable controlled- or sustained-release microneedle tip cancomprise less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%or more of the total dosage amount.E63. The device of embodiment E59-E61, wherein the implantablemicroneedle tip comprises about 0.1 μg to about 65 μg of vaccine (e.g.,about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg,about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1 μg,about 1 μg to about 10 μg, about 10 μg to about 20 μg, about 20 μg toabout 30 μg, about 30 μg to about 40 μg, about 40 μg to about 50 μg,about 50 μg to about 65 μg of a vaccine, antigen, and/or immunogendescribed herein).E64. A method of providing immunity to a virus, e.g., broad spectrumimmunity, in a subject comprising contacting the skin of the subjectwith the microneedle of any one of embodiments E1-E53, or the device ofany one of embodiments E58-E63.E65. A method of providing a controlled- or sustained-release of avaccine, e.g., an influenza vaccine, in a subject comprising contactingthe skin of the subject with the microneedle of any one of embodimentsE1-E53, or the device of any one of embodiments E58-E63.E66. A method of enhancing an immune response to a virus, e.g., aninfluenza virus, in a subject comprising contacting the skin of thesubject with a microneedle of any one of embodiments E1-E53, or thedevice of any one of embodiments E58-E63.E67. The method of any one of embodiments E64-E66, wherein theimplantable sustained-release tip is configured to release a vaccineinto the skin of the subject over a period of time comprising at leastabout 4 days (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 or more days, e.g., between about 4 days and about 14 days, e.g.,between about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks).E68. The method of embodiment E67, wherein the implantablesustained-release tip is configured to release a vaccine into the skinof the subject over a period of time comprising between about 1 week toabout 2 weeks (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days).E69. The method of any one of embodiments E64-E68, wherein immuneresponse is a cellular and/or humoral immune response comprising:

(i) an elevated hemagglutination inhibition (HAI) antibody titerdetectable in the blood of the subject, e.g., detectable at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 51, and/or 52-weeks or more postimmunization, optionally wherein the elevated HAI antibody titer is to adrifted influenza A, B, C, and/or D strain;

(ii) an elevated anti-influenza IgG titer detectable in the blood of thesubject, e.g., detectable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,and/or 12-months or more post immunization, optionally wherein theelevated anti-influenza IgG titer is to a drifted influenza A, B, C,and/or D strain; and/or

(iii) a level of antibody secreting plasma cells (ASC) against thevirus, e.g., the influenza virus, e.g., the drifted influenza A, B, C,and/or D strain, detectable in the bone marrow of the subject, e.g.,detectable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51,and/or 52-weeks or more post immunization.

E70. The method of embodiment E69, wherein:

(i) an elevated hemagglutination inhibition (HAI) antibody titer isdetectable in the blood of the subject for the duration of a completeflu season post immunization, optionally wherein the elevated HAIantibody titer is to a drifted influenza A, B, C, and/or D strain;and/or

(ii) the percent seroconversion, e.g., based on the elevated HAIantibody titer detectable in the blood of the subject, e.g., at 6-monthpost immunization is greater than about 20% (e.g., 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more,e.g., 100%).

E71. The method of embodiment E69 or E70, wherein:

(i) the immune response is a cellular immune response comprising anincrease in the level of IFNγ secreting cell in the blood of thesubject, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51,and/or 52-weeks or more post immunization; And/or

(ii) the elevated HAI antibody titer, the elevated anti-influenza IgGtiter, the level of antibody secreting plasma cells (ASC) against thevirus, and/or the level of IFNγ secreting cells detectable in thesubject is greater (e.g., 1-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold,14-fold, or 15-fold or more greater) as compared to the administrationof a single-dose or a bolus administration of the vaccine.

E72. A method of producing a microneedle device, the method comprising:

providing a mold including a mold body with an array of needle cavitieshaving a predefined shape, e.g., pyramid-shaped and/or conical-shapedneedle cavities, formed therein;

filling tips of the needle cavities with a composition consisting of asilk fibroin, antigen solution;

drying the filled tips of the needle cavities to create releasable tips,and optionally annealing the needle tips;

filling the needle cavities of the mold with a dissolvable basesolution;

drying the dissolvable base solution to create base layers for thereleasable tips; and

applying a backing layer to the base layers to create a microneedledevice.

E73. The method of embodiment E72, further comprising removing themicroneedle device from the mold.E74. The method of embodiment E73, wherein the microneedle device isremoved by bending the mold away from the microneedle device.E75. The method of embodiment E73, further comprising packagingmicroneedle devices in a container with low moisture vapor transmissionrate with a desiccant to maintain between about 0% and about 50% (e.g.,between about 0% and 10%, between about 10% and about 20%, between about20% and about 30%, between about 30% and about 40%, or between about 40%and 50%, e.g., about 25%) relative humidity inside the package.E76. The method of embodiment E72, wherein the silk fibroin, antigensolution is dispensed into each needle cavity in the mold via nanoliterprinting.E77. The method of embodiment E76, wherein filling the tips of theneedle cavities includes dispensing a solution, e.g., an antigen-silkformulation into each needle cavity.E78. The method of embodiment E72, wherein drying the filled tips of theneedle cavities includes a primary drying step and a secondary dryingstep.E79. The method of embodiment E72, wherein filling the needle cavitiesof the mold with a dissolvable base solution includes a solution of 40%w/v Hydrolyzed Gelatin and 10% w/v Sucrose in deionized water (DIW).E80. The method of embodiment E79, wherein filling the dissolvable basesolution includes subjecting the mold to a centrifuge at 3900 rpm for 2minutes and topping off the needle cavities with 50 μL of base solution.E81. The method of embodiment E62, further comprising an annealing step(e.g., before filling the base) after the filling the tips of the needlecavities.E82. The method of embodiment E62, further comprising a water annealingstep (e.g., before filling the base) after the filling the tips of theneedle cavitiesE83. The method of embodiment E62, wherein the backing layer includesone of a paper backing layer and an adhesive plastic tape.E84. The use of a microneedle of any one of embodiments E0-E53 in amethod of providing immunity to a virus, e.g., an influenza virus.E85. The use of a microneedle of any one of embodiments E0-E53 in amethod of providing a controlled- or sustained-release of a vaccine,e.g., an influenza vaccine, in a subject.E86. The use of a microneedle of any one of embodiments E0-E53 in amethod of enhancing an immune response to a virus, e.g., an influenzavirus, in a subject.E87. The microneedle of any one of embodiments E0-E53, for use as amedicament, e.g., in any of the method embodiments described herein.E88. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises between about 1% and about 75% (e.g.,about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about30%, or about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, or about 75% hyaluronate).E89. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises between about 1% and about 75% (e.g.,about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about30%, or about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, or about 75% maltose).E90. The microneedle of any one of the preceding embodiments, whereinthe dissolvable comprises between about 1% and about 75% (e.g., about1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, orabout 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, or about 75% methyl cellulose).E91. The microneedle of embodiment E1 or E2, wherein the dissolvablebase comprising eight of gelatin, PEG, sucrose, CMC, PVP, PVA,hyaluronate, maltose, and methyl cellulose.E92. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises PEG.E93. The microneedle of any one of the preceding embodiments, whereinthe dissolvable base comprises between about 1% and about 70% PEG (e.g.,about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about50%, about 60%, or about 70% PEG).E94. A method for providing broad-spectrum immunity to a virus, e.g., aninfluenza virus, in a subject, said method comprising administering avaccine (e.g., a influenza vaccine) in an amount (e.g., a dosage) and/orover a time period sufficient to result in broad-spectrum immunity to avirus, e.g., results in an immune response (e.g., a cellular immuneresponse and/or a humoral immune response) to a drifted strain of thevirus, in the subject.E95. The method of embodiment E94, wherein the vaccine is administeredin a composition for the controlled- or sustained-release of the vaccine(e.g., for the controlled- or sustained-release of one or more viralantigens as described herein).E96. The method of embodiment E94, wherein the vaccine is administeredby a device for the controlled- or sustained-release of the vaccine(e.g., for the controlled- or sustained-release of one or more viralantigens as described herein).E97. The method of any of embodiments E94-96, wherein the vaccine isadministered into a subject, e.g., in to a tissue or cavity of thesubject chosen from skin, mucosa, organ tissue, muscle tissue or buccalcavity.E98. The method of any of embodiments E94-E97, wherein the vaccine isadministered in an amount (e.g., a dosage) and/or over a time periodsufficient to result in one or more of:

(i) exposure in the subject to one or more antigens in the vaccine in anamount and/or period of time to result in broad spectrum immunity, e.g.,to result in an immune response (e.g., a cellular immune response and/ora humoral immune response) to a drifted strain of the virus, in thesubject; or

(ii) a level of one or more antigens in the subject that issubstantially steady, e.g., about 20%, 15%, 10%, 5%, or 1% to an amount,e.g., minimum amount, needed to result in an immune response (e.g., acellular immune response and/or a humoral immune response) to the one ormore antigens.

E99. The method of any one of embodiments E95-98, wherein thecomposition or device for the controlled- or sustained-release of thevaccine is chosen from: a microneedle (e.g., a microneedle device, e.g.,a microneedle patch), an implantable device (e.g., a pump, e.g., asubcutaneous pump), an injectable formulation, a depot, a gel (e.g., ahydrogel), an implant, or a particle (e.g., a microparticle and/or ananoparticle).E100. The method of embodiment E99, wherein the device for thecontrolled- or sustained-release of the vaccine comprises a microneedleor microneedle device, e.g., described herein.E101. The method of embodiment E99, wherein the device for thecontrolled- or sustained-release of the vaccine comprises a pump (e.g.,a subcutaneous pump).E102. The method of embodiment E99, wherein the composition for thecontrolled- or sustained-release of the vaccine comprises an injectableformulation (e.g., an injectable depot formulation).E103. The method of embodiment E100, wherein the composition for thecontrolled- or sustained-release of the vaccine comprises an implant.E104. The method of embodiment E100, wherein the composition for thecontrolled- or sustained-release of the vaccine comprises a gel (e.g., ahydrogel).E105. The method of any one of embodiments E100-E104, wherein thecomposition or device for the controlled- or sustained-release of thevaccine comprises a particle (e.g., a microparticle and/or ananoparticle).E106. The method of any one of embodiments E94-104, wherein the vaccineis administered, e.g., released by the composition or device for thecontrolled- or sustained-release of the vaccine, e.g., into the subject,in order to maintain a vaccine dosage (e.g., an antigen concentration)for a period of time sufficient to result in broad spectrum immunity,e.g., to result in an immune response (e.g., a cellular immune responseand/or a humoral immune response) to a drifted strain of the virus, inthe subject (e.g., wherein the period of time is about 1 to 21 days,e.g., about 5 to 10 days or about 5 to 7 days, e.g., about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days).E107. The method of embodiment E106, wherein the composition or devicefor the controlled- or sustained-release of the vaccine maintainsantigen release and/or level in the subject over a sustained period oftime.E108. The method of embodiment E106, wherein the composition or devicefor the controlled- or sustained-release of the vaccine maintains acontinuous or non-continuous antigen release into the subject over asustained period of time.E109. The method of any one of embodiments E94-E108, wherein the vaccineis administered, e.g., released by the composition or device for thecontrolled- or sustained-release, over a period of time comprising atleast about one week, e.g., about 1-2 weeks, about 1-3 weeks, or about1-4 weeks.E110. The method of any one of embodiments E94-E109, wherein the vaccineis administered, e.g., released by the composition or device for thecontrolled- or sustained-release, over a period of time comprising atleast about 4 days (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 days, or more, e.g., between about 4 days andabout 2 weeks, between about 4 days and about 1 week).E111. The method of any one of embodiments E94-E111, wherein the vaccineis administered in a dosage comprising between about 0.1 μg and about 65μg per strain, e.g., 0.2 μg and about 50 μg per strain (e.g., about eachof 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, or 65 μg per strain).E112. The method of any one of embodiments E94-E111, wherein at leastabout 1% of the dosage of the vaccine (e.g., at least about 0.5% toabout 10%, at least about 5% to about 15% at least about 10% to about20% of the dosage), e.g., released by the composition or device for thecontrolled- or sustained-release of the vaccine, e.g., into the subject,is maintained over a period of time comprising at least about 4 days(e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or more,e.g., between about 4 days and about 2 weeks, between about 4 days andabout 1 week).E113. The method of any one of embodiments E94-E112, wherein the vaccineis administered, e.g., released by the composition or device for thecontrolled- or sustained-release, in a plurality of fractional doses ofa total dose (e.g., a standard dose) over a time period, e.g., such thatan immune response and/or broad-spectrum immunity is achieved, whereinthe amount of the vaccine administered in each of the fractional dosesis no more than 1/X, wherein X is any number, e.g., wherein X is 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more, of the total dose(e.g., a standard dose) of the vaccine.E114. The method of any one of embodiments E94-E112, wherein the vaccineis administered, e.g., released by the composition or device for thecontrolled- or sustained-release of the vaccine, e.g., into the skin ofthe subject, in a plurality of doses equivalent to a percentage of atotal dose (e.g., a percentage of a standard dose) over a time period,e.g., such that broad-spectrum immunity is achieved,

wherein the amount of the vaccine administered in each of the pluralityof doses is about X %, wherein X is any number, e.g., wherein X is 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 300, 400,or 500 or more, of the total dose (e.g., a standard dose) of thevaccine.

E115. The method of embodiment E113 or E114, wherein the vaccine isadministered such that broad-spectrum immunity is achieved, e.g., suchthat an immune response, e.g., a cellular immune and/or humoral immuneresponse to a drifted strain is achieved.E116. The method of embodiment E113 or E114, wherein the vaccine isadministered as two, three, four, five, six, seven, eight, nine, ten ormore fractional doses.E117. The method of any one of embodiments E113-116, wherein the totaldose (e.g., the standard dose) of the vaccine is administered to achievebroad-spectrum immunity.E118. The method of any one of embodiments E113-116, wherein less thanthe total dose (e.g., the standard dose) of the vaccine is administeredto achieve broad-spectrum immunity.E119. The method of any one of embodiments E113-116, wherein more thanthe total dose (e.g., the standard dose) of the vaccine is administeredto achieve broad-spectrum immunity.E120. The method of any one of embodiments E113-116, wherein the amountof the vaccine administered in each of the fractional doses is the same.E121. The method of any one of embodiments E113-116, wherein the amountof the vaccine administered in each of the fractional doses isdifferent.E122. The method of any one of embodiments E113-121, wherein theplurality of fractional doses is administered by intramuscular injectionor intradermal injection, e.g., to achieve controlled- orsustained-release of a vaccine.E122. The method of any one of embodiments E113-E122, wherein each doseof the plurality of fractional doses is administered at least once ortwice a day, at least once every two days, at least once every threedays, at least once every four days, at least once every five days, atleast once every 6 days, at least one a week, or at least once a monthfor the duration of the time period.E124. The method of any one of embodiments E94-E123, wherein:

(i) the vaccine comprises a first influenza strain and administration ofa dose of the first influenza strain to the subject results inbroad-spectrum immunity to a second influenza strain (e.g., a driftedinfluenza strain) not present in the implantable sustained-release tipor the vaccine;

(ii) the vaccine comprises a first influenza A strain and administrationof a dose of the first influenza A strain to the subject results inbroad-spectrum immunity to a drifted influenza strain (e.g., a driftedinfluenza A, B, C, and/or D strain) not present in the implantablesustained-release tip or the vaccine;

(iii) the vaccine comprises a first influenza B strain andadministration of a dose of the first influenza B strain to the subjectresults in broad-spectrum immunity to a drifted influenza strain (e.g.,a drifted influenza A, B, C, and/or D strain) not present in theimplantable sustained-release tip or the vaccine;

(iv) the vaccine comprises a first influenza C strain and administrationof a dose of the first influenza C strain to the subject results inbroad-spectrum immunity to a drifted influenza strain (e.g., a driftedinfluenza A, B, C, and/or D strain) not present in the implantablesustained-release tip or the vaccine; and/or

(v) the vaccine comprises a first influenza D strain and administrationof a dose of the first influenza D strain to the subject results inbroad-spectrum immunity to a drifted influenza strain (e.g., a driftedinfluenza A, B, C, and/or D strain) not present in the implantablesustained-release tip or the vaccine.

E125. The method of embodiment E124, wherein the first influenza Avaccine comprises:

(i) an H1N1 (e.g., A/Michigan and/or A/California) vaccine; and/or

(ii) an H3N2 (e.g., A/Hong Kong and/or A/Switzerland) vaccine.

E126. The method of embodiment E124 or E125, wherein the driftedinfluenza A strain comprises:

(i) an H1N1 strain (e.g., A/Michigan and/or A/California); and/or

(ii) an H3N2 strain (e.g., A/Hong Kong and/or A/Switzerland).

E127. The method of any one of embodiments E124-E126, wherein:

(i) the first influenza A vaccine comprises an H1N1 vaccine toA/Michigan and the drifted influenza A strain comprises A/California;and/or

(ii) the first influenza A vaccine comprises an H3N2 vaccine to A/HongKong and the drifted influenza A strain is A/Switzerland.

E128. The method of embodiment E124, wherein the first influenza Bvaccine comprises:

(i) a B/Yamagata lineage strain (e.g., B/Phuket); and/or

(ii) a B/Victoria lineage strain (e.g., B/Brisbane).

E129. The method of embodiment E124 or E128, wherein:

(i) the drifted influenza B strain is a B/Yamagata lineage strain (e.g.,B/Phuket); and/or

(ii) the drifted influenza B strain is a B/Victoria lineage strain(e.g., B/Brisbane).

E130. The method of any one of embodiments E124, E128, or E129, whereinthe first influenza B vaccine is to the B/Victoria lineage strainB/Brisbane and the drifted influenza B strain is the B/Yamagata lineagestrain B/Phuket.E131. The method of any one of embodiments E94-E130, wherein the immuneresponse and/or broad-spectrum immunity comprises a cellular and/orhumoral immune response comprising:

(i) an elevated hemagglutination inhibition (HAI) antibody titerdetectable in the blood of the subject, e.g., detectable at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 51, and/or 52-weeks or more postimmunization, optionally wherein the elevated HAI antibody titer is to adrifted influenza A, B, C, and/or D strain;

(ii) an elevated anti-influenza IgG titer detectable in the blood of thesubject, e.g., detectable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,and/or 12-months or more post immunization, optionally wherein theelevated anti-influenza IgG titer is to a drifted influenza A, B, C,and/or D strain; and/or

(iii) a level of antibody secreting plasma cells (ASC) against thevirus, e.g., the influenza virus, e.g., the drifted influenza A, B, C,and/or D strain, detectable in the bone marrow of the subject, e.g.,detectable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51,and/or 52-weeks or more post immunization.

E132. The method of embodiment E131, wherein an elevatedhemagglutination inhibition (HAI) antibody titer is detectable in theblood of the subject for the duration of a complete flu season postimmunization, optionally wherein the elevated HAI antibody titer is to adrifted influenza A, B, C, and/or D strain.E133. The method of embodiment E131, wherein the percent seroconversion,e.g., based on the elevated HAI antibody titer detectable in the bloodof the subject, e.g., at 6-month post immunization is greater than about20% (e.g., 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% or more, e.g., 100%).E134. The method of any one of embodiments E94-E133, whereinbroad-spectrum immunity comprises a cellular immune response comprisingan increase in the level of IFNγ secreting cell in the blood of thesubject, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51,and/or 52-weeks or more post immunization.E135. The method of any one of embodiments E111-E133, wherein theelevated HAI antibody titer, the elevated anti-influenza IgG titer, thelevel of antibody secreting plasma cells (ASC) against the virus, and/orthe level of IFNγ secreting cells detectable in the subject is greater(e.g., 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold or moregreater) as compared to the administration of a single-dose or a bolusadministration of the vaccine.E136. A method for providing an immune response (e.g., a cellular immuneresponse and/or a humoral immune response) and/or a broad spectrumimmunity to a virus, e.g., an influenza virus, in a subject, said methodcomprising administering a vaccine (e.g., a influenza vaccine) in anamount (e.g., a dosage) and/or over a time period sufficient to elicitan immune response (e.g., a cellular immune response and/or a humoralimmune response) to the virus, e.g., the influenza virus, in thesubject,

wherein the vaccine is administered in a composition for the controlled-or sustained-release of the vaccine (e.g., for the controlled- orsustained-release of one or more viral antigens as described herein)over a period of time comprising about 1 to about 2 weeks (e.g., about10 days).

E137. A method of the any of the preceding embodiments, wherein thesubject (e.g., the human subject) is a pediatric subject.E138. A method of the any of the preceding embodiments, wherein thesubject (e.g., the human subject) is an adult subject.E139. A method of the any of the preceding embodiments, wherein thesubject (e.g., the human subject) is an elderly subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1F are a series of graphs showing that the sustainedintradermal delivery of an influenza vaccine generates improved cellularresponses and stronger, longer-lasting antibody responses. Balb/c mice(n=5/group) were immunized with Fluzone HD® vaccine by intramuscularinjection (IM) or the same dose injected intradermally as fractionaldoses over 10 days (SR or ID Sus. Rel.) or were unimmunized (Naïve).Anti-flu vaccine IgG titers were measured by ELISA over 5 months postimmunization (FIGS. 1A-1B), hemagglutination inhibition titers measuredusing Turkey RBCs at days 28 and 56 (FIGS. 1C and 1D), and vaccinespecific IFNγ+ cells in peripheral blood determined by ELISPOT at week12 post immunization with representative images above the graph (FIGS.1E and 1F). Data are presented as mean±SEM, n=5/group, n.s notsignificant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 with two-wayANOVA and Tukey's post test in A, two way ANOVA and Sidak's post test inB.

FIGS. 2A-2J are a series of graphs showing that immunization viacontrolled- or sustained-release silk microneedles improves humoral andcellular responses. Balb/c mice (n=5/group) were immunized with FluzoneHD® vaccine by either intramuscular injections (IM) or microneedles (MN)that can sustain release of the vaccine in the skin, or were unimmunized(Naïve). Following immunization, the anti-flu IgG titers were measuredby ELISA (FIGS. 2A-2B). As shown in FIG. 2B, a 3-5 fold increase intiters is observed for 6 months post immunization with MN compared to IMinjection. HAI titers for the 3 strains, A/Hong Kong/H3N2,A/Michigan/H1N1 and B/Brisbane were measured at months 1, 2, 3, 4 and 6post immunization (FIGS. 2C-2H). Significantly higher HAI titers wereobserved with MN with complete seroconversion maintained at month 6compared to IM injection for the two A strains and a trend towardsimproved seroconversion for the B lineage (FIGS. 2D, 2F, and 2H). IFNγcellular responses in peripheral blood was also significantly higherupon MN delivery of vaccine than IM delivery (FIGS. 2E-2F). At week 4post vaccination, significantly higher vaccine specific IFNγ+ cells inperipheral blood was determined by ELISPOT for MN delivery withrepresentative images shown above the graph (FIGS. 2I-2J). These resultsdemonstrate the enhanced immunogenicity of vaccination possible thoughmicroneedle delivery. Data in are presented as mean±SEM, n=5/group,*p<0.01, **p<0.01, ***p<0.001, with two-way ANOVA and Tukey's post testin FIG. 2B, two way ANOVA and Sidak's post test in FIGS. 2D, 2F, and 2H,one way ANOVA with Tukey's post test in FIG. 2J. Two way ANOVA withTukey's post test for 2C, 2E, and 2G, One-way ANOVA with Tukey's posttest for 2I. *p<0.05, ***p<0.001, **** p<0.0001.

FIG. 2K illustrates an enlarged view of a portion of a fabricatedmicroneedle device (top panel) prior to and after application to theskin. As shown, the tips of the needles are distinct from theirrespective bases, and comprise fluorescently labelled silk (Silk-AF568)and antigen (antigen-AF647). The bottom panel shows that antigen releasecan be extended to at least six days compared to equivalent injection inmice. Without being bound by theory, the limit-of-detection for IVISimaging (whole animal) is approximately <1% of full dose (e.g., standarddose). In some embodiments, after application of a microneedle loss ofsignal is measured by IVIS imaging at about 6-7 days post immunization.

FIG. 3 is a schematic drawing of the microneedle fabrication process.

FIG. 4 illustrates a completed microneedle device having an array ofmicroneedles applied to a backing or “handle” layer.

FIG. 5 illustrates various molecular weight profiles of silk fibroinsolutions useful in fabricating a microneedle described herein.

FIGS. 6A-6B are a series of graphs showing that controlled- orsustained-release of influenza vaccine generates higher HAI titersagainst drifted H3N2 strain of influenza. Balb/c mice were immunizedwith FluzoneHD at 0.5 ug/strain either by intramuscular injection (IM,gray circle) or by intradermal injections of fractional doses for atotal of 10 days (SR, black diamond) or by application of the MIMIXmicroneedle patch (MN, black squares). Naïve mice are indicated by opentriangles. HAI titers for A/Switzerland/H3N2/2013 (a strain that was notincluded in the vaccine) were measured at month 4 and 5 (days 120 and150) post immunization respectively. As shown in the figure, 10 daycontrolled- or sustained-release of vaccine (SR) results insignificantly higher titers to the drifted strain compared to equivalentintramuscular injections. Haemagglutination inhibition titers above 40are known correlates of protection against infection. MIMIX (MN)delivery also showed a trend towards increased HAI titers with 3 out of5 mice achieving a HAI titer of 40 compared to no animals in the IMimmunized group at month 4 post immunization indicating highercorrelates of protection by controlled- or sustained-release. Data arepresented as mean±SEM, n=5/group, *p<0.05, ****p<0.0001 with one-wayANOVA and Tukey's post test in A and B. Dotted line indicatesseroconversion.

FIGS. 7A-7B are a series of graphs showing that controlled- orsustained-release of influenza vaccine generates more long-lived plasmacells in the bone marrow against both vaccine included and drifted H3N2strains of influenza. Balb/c mice were immunized with FluzoneHD at 0.5ug/strain either by intramuscular injection (IM, gray bar) or byintradermal injections of fractional doses for a total of 10 days (SR,black bar). At month 8 (day 240) post immunization, animals weresacrificed and the cells from the bone marrow were isolated. A B cellELISPOT was performed (following manufacturer's instructions,Immunospot) to measure antibody secreting plasma cells (ASC) against thevaccine included strain (A/Hong Kong/H3N2) and drifted strain(A/Switzerland/H3N2). Data demonstrates that fractional dosing of thevaccine over 10 days (SR) resulted in significantly higher number ofboth vaccine-specific and drifted strain specific ASCs withrepresentative images above the graphs. Data are presented as mean±SEM,n=5/group, *p<0.05, one-way ANOVA and Tukey's post test in A and B.

FIG. 8 is a graph showing that controlled- or sustained-release ofinfluenza vaccine generates higher HAI titers against drifted H1N1strain of influenza. Balb/c mice were immunized with FluzoneHD at 0.5ug/strain either by intramuscular injection (IM, gray circle), byintradermal injections of fractional doses for a total of 10 days (SR,black diamond) or by application of the MIMIX patch (MN, black square).Naïve mice are indicated by open triangles. HAI titers forA/California/7/2009/H1N1 (a strain that was not included in the vaccine)were measured at month 6 (day 180) post immunization. As shown in thefigure, 10 day controlled- or sustained-release of vaccine (SR) resultsin significantly higher titers compared to equivalent intramuscularinjections to the drifted vaccine strain. Haemagglutination inhibitiontiters above 40 are known correlates of protection against infection.MIMIX (MN) delivery also showed a trend towards increased HAI titerswith 3 out of 5 mice achieving a HAI titer of 40 compared to 1 animalresponding in the IM immunized group indicating higher correlates ofprotection by controlled- or sustained-release. Data are presented asmean±SEM, n=5/group, n.s. not significant, *p<0.05, **p,0.01, one-wayANOVA and Tukey's post test.

FIGS. 9A-9B are a series of graphs showing that controlled- orsustained-release of influenza vaccine generates more long-lived plasmacells in the bone marrow against both vaccine included and drifted H1N1strains of influenza. Balb/c mice were immunized with FluzoneHD at 0.5ug/strain either by intramuscular injection (IM, gray bar) or byintradermal injections of fractional doses for a total of 10 days (SR,black bar). At month 8 (day 240) post immunization, animals weresacrificed and the cells from the bone marrow were isolated. A B cellELISPOT was performed (following manufacturer's instructions,Immunospot) to measure antibody secreting plasma cells (ASC) against thevaccine included strain (A/Michigan/H1N1) and not included driftedstrain (A/California/H1N1). As shown in the figure, fractional dosing ofthe vaccine over 10 days (SR) showed a trend towards increase in bothvaccine-specific and drifted strain specific ASCs with representativeELISPOT images above the graphs. Data are presented as mean±SEM,n=5/group.

FIG. 10 is a graph showing that controlled- or sustained-release ofinfluenza vaccine generates higher HAI titers against B lineage notincluded in the vaccine. Balb/c mice were immunized with FluzoneHD at0.5 ug/strain either by intramuscular injection (IM, gray bar) or byapplication of the MIMIX patch (MIMIX, black bar). HAI titers forB/Phuket were measured at week 7 (day 49) post immunization. B/Phuketbelongs to the Yamagata lineage that was not included in the vaccine. Asshown in the figure, sustained vaccine release from MIMIX showed a trendtowards increase in HAI titers to this B lineage.

FIGS. 11A-11B are a series of graphs showing that controlled- orsustained-release of influenza vaccine generates more long-lived plasmacells in the bone marrow against both vaccine included and non includedB lineages of influenza. Balb/c mice were immunized with FluzoneHD at0.5 ug/strain either by intramuscular injection (IM, gray bar) or byintradermal injections of fractional doses for a total of 10 days (SR,black bar). At month 8 (day 240) post immunization, animals weresacrificed and the cells from the bone marrow were isolated. A B cellELISPOT was performed (following manufacturer's instructions,Immunospot) to measure antibody secreting plasma cells (ASC) against thevaccine included B/lineage strain (B/Brisbane) and to the B/Yamagatalineage (B/Phuket). As shown in the figure, fractional dosing of thevaccine over 10 days (SR) resulted in significantly higher number ofboth vaccine-specific and drifted strain specific ASCs. Data arepresented as mean±SEM, n=5/group, *p<0.05, **p<0.01, one-way ANOVA andTukey's post test in A and B.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery thatmodulating the kinetics of antigen presentation to mimic that of anatural infection (e.g., a viral infection) can drive a more potentimmune response (e.g., a more potent cellular and/or humoral immuneresponse) (see, e.g., Tam et al. PNAS. 113:E6639-E6648, 2016; andSchipper at al. J. Control Release. 242:141-147, 2016). Without wishingto be bound by theory, the microneedles and microneedle devicesdescribed herein can mimic the natural process of antigen presentation(e.g., viral antigen presentation) by enabling the release, e.g.,controlled- or sustained-release, of a virus-derived antigen, immunogen,and/or vaccine into a subject, e.g., into the dermis skin layer of asubject. The controlled- or sustained-release enabled by theformulations, compositions, articles, devices, and preparations,microneedles, and microneedle devices described herein can inducegreater immunogenicity, an enhanced immune response (e.g., a more potentcellular and/or humoral immune response), and/or broad-spectrum immunityin a subject, as compared to the administration of single-dose or bolusadministration of, e.g., a vaccine, such as an influenza vaccine.

In some embodiments, the microneedles and microneedle devices describedherein can comprise an implantable controlled- or sustained-releasesilk-based microneedle tip that encapsulates and/or stabilizes atherapeutic agent, such as a vaccine, an antigen, and/or an immunogen(e.g., an influenza vaccine); and a dissolving base layer that supportsthe distal microneedle tip. Upon application of a microneedle ormicroneedle device, as described herein, to a biological barrier of asubject, the base layer dissolves and the silk-based microneedle tipsare implanted at a predetermined depth (e.g., a max penetration depth ofthe distal part of tip) within the biological barrier (e.g., the dermislayer of the skin, e.g., at a depth of between about 100 μm and about800 μm). In some embodiments, the whole tip is not embedded within,e.g., the dermis layer of the skin, e.g., at a depth of between about100 μm and about 800 μm. The implanted tip then slowly releases thetherapeutic agent over a time period sufficiently long enough to enableimmunity (e.g., over a time period of at least about 4 days (e.g., about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or more days, e.g.,between about 4 days and about 14 days, e.g., between about 1-2 weeks,about 1-3 weeks, or about 1-4 weeks, e.g., about one week, about twoweeks, about three weeks, about four weeks, about five weeks, or aboutsix weeks or more weeks). Various properties of the silk fibroin matrixcomprising the implantable controlled- or sustained-release microneedletip, including, for example, crystallinity, beta-sheet content, andmolecular weight, can be modulated to tune (e.g., alter and/or modify)the release kinetics (e.g., rate of release) of a therapeutic agent,such as a vaccine, an antigen, and/or an immunogen from the microneedletip. In some embodiments, the implantable controlled- orsustained-release microneedle tip comprises a beta-sheet content ofbetween about 10% and about 60% (e.g., about 10%, about 20%, about 30%,about 40%, about 50%, about 60%), e.g., as based on a “crystallinityindex,” e.g., a “crystallinity index” known in the art.

In some embodiments, the controlled- or sustained-release formulations,compositions, articles, devices, and preparations, comprise at least onetherapeutic agent, e.g., at least one vaccine, antigen, and/or immunogendescribed herein. In some embodiments, the formulations, compositions,articles, devices, and preparations for controlled- and/or sustainedrelease described herein release a therapeutic agent (e.g., a vaccine)over a time period sufficiently long enough to enable immunity (e.g.,over a time period of at least about 1 to about 14 days (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or more days, e.g.,between about 4 days and about 14 days, e.g., between about 1-2 weeks,about 1-3 weeks, or about 1-4 weeks, e.g., about one week, about twoweeks, about three weeks, about four weeks, about five weeks, or aboutsix weeks or more weeks). Accordingly controlled- or sustained-releaseformulations, compositions, articles, devices, and preparations,microneedles, microneedle devices, kits, as well as methods of makingand using the same are disclosed.

Definitions

All scientific and technical terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. References to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques or substitutions ofequivalent or later-developed techniques which would be apparent to oneof skill in the art. In addition, in order to more clearly and conciselydescribe the subject matter which is the invention, the followingdefinitions are provided for certain terms which are used in thespecification and appended claims.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “about” means+/−10% of the recited value.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.”

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

As used herein, an “adjuvant” is a substance that is able to favor oramplify the cascade of immunological events, ultimately leading to anincreased immunological response, e.g., the integrated bodily responseto an antigen, including cellular and/or humoral immune responses.Non-limiting examples of adjuvants include: aluminum (e.g., aluminumgels and/or aluminum salts, such as aluminum hydroxide, aluminumphosphate, and aluminum potassium sulfate), lipids (e.g., squalene,monophosphoryl lipid A (MPL)), AS03 (e.g., an adjuvant comprisingD,L-alpha-tocopherol (vitamin E), squalene, and polysorbate 80), AS04(e.g., an adjuvant comprising a combination of aluminum hydroxide andMPL), and MF59® (e.g., an adjuvant comprising squalene).

As used herein, the term “antigen” refers to refers to a moleculecapable of inducing a humoral immune response and/or cellular immuneresponse, e.g., leading to the activation of B and/or T lymphocytesand/or innate immune cells and/or antigen presenting cells. Anymacromolecule, including almost all proteins or peptides, can be anantigen. Antigens can also be derived from genomic and/or recombinantDNA. For example, any DNA comprising a nucleotide sequence or a partialnucleotide sequence that encodes a protein capable of eliciting animmune response encodes an “antigen.” In some embodiments, an antigendoes not need to be encoded solely by a full length nucleotide sequenceof a gene, nor does an antigen need to be encoded by a gene at all. Insome embodiments, an antigen can be synthesized or can be derived from abiological sample, e.g., a tissue sample, a tumor sample, a cell, or afluid with other biological components. In some embodiments, an antigencan be derived from a virus. Antigens as used herein may also bemixtures of several individual antigens.

As used herein, the term “backing” refers to a material that is suitablefor bonding to and/or adhering to a component of a microneedle. In someembodiments, a backing material is suitable for bonding to and/oradhering to the dissolvable base of a microneedle described herein.

As used herein, the phrase “broad-spectrum immunity” refers to an immuneresponse, e.g., a humoral and/or cellular response (e.g., immunity orprotective immunity), against at least one (e.g., against at least two,at least three, at least four, at least five, against at least eight, orat least against more than eight) strains of a virus (e.g., a virusdescribed herein), wherein the at least one strain is not present in avaccine administered to a subject, e.g., according to the methods,microneedles, and microneedle devices described herein. In someembodiments, the at least one strain not present in the vaccine is adrifted strain of the virus. In some embodiments, the at least onestrain belongs to a different type as the strain(s) present in thevaccine.

As used herein, the term “immunity” or “protective immunity” refers toan immune response, e.g., a humoral and/or cellular response, elicitedby a vaccine or immunization schedule (e.g., vaccination regimen) thatwhen administered to a subject in need thereof (e.g., a subjectdescribed herein), that prevents, retards the development of, and/orreduces the severity of a viral infection that is caused by a virusdescribed herein. In some embodiments, immunity or protective immunitydiminishes or altogether eliminates the symptoms of the viral infection.In some embodiments, immunity or protective immunity is characterized bythe presence of one or more of: circulating antibodies (e.g., humoralimmunity), the presence of sensitized T lymphocytes (e.g., cellularimmunity), the presence of secretory IgA on mucosal surfaces (e.g.,mucosal immunity), or a combination thereof.

As used herein, the term “antigenic drift” refers to a mutation in thegene of an influenza virus that accumulates over time as the virusreplicates. These mutations usually produce viruses that are closelyrelated to one another (e.g., located close together on a phylogenetictree), and referred to herein as “drifted strains.” In some embodiments,viruses that are closely related to each other share similar antigenicproperties and an immune system exposed to a first virus and,subsequently, a drifted strain of the first virus will usually recognizethe drifted strain and respond to it by mounting an immune response(e.g., a protective immune response), referred to as “cross-protection.”However, in some embodiments these small genetic changes can accumulateover time and result in viruses that are antigenically different (e.g.,located further away on a phylogenetic tree), and when this happens, thebody's immune system may not recognize those viruses (e.g., thosedrifted strains).

As used herein, the term “dissolvable base” refers to the layer thatforms the base of the microneedles (e.g., functions as the support forthe distal implantable silk tips that are loaded with a vaccine, anantigen, or an immunogen), and/or can also serve as a layer connectingadjacent microneedles to form a continuous microneedle array ormicroneedle patch. In some embodiments, at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or more of the base is dissolved afterapplication to a biological barrier, e.g., skin or mucous surface, orbuccal cavity.

As used herein, the term “dose” means the amount of a vaccine, antigen,and/or immunogen which is administered (e.g., in a vaccination) toelicit an immune response (e.g., a humoral and/or a cellular immuneresponse) in an organism.

As used herein, a “standard dose” means the amount of antigen in atypical human dose of a vaccine, as approved for marketing by nationalor international regulatory authorities (e.g., U.S. FDA, EMEA).

As used herein, a “fractional dose” refers to a dosage comprising aportioned amount of a total dose (e.g., a standard dose) of a vaccine,antigen, and/or immunogen which is administered (e.g., in a vaccination)to elicit an immune response (e.g., a humoral immune response, acellular immune response, and/or a broad-spectrum immunity) in anorganism. In some embodiments, the amount of the vaccine, antigen,and/or immunogen in the fractional dose is no more than 1/X, wherein Xis any number, e.g., wherein X 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90,or 100 or more, of the total dose (e.g., a standard dose) of thevaccine.

As used herein, the term “gelatin” refers to a water-soluble proteinderived from collagen. In some embodiments, the term “gelatin” refers toa sterile nonpyrogenic protein preparation (e.g., fractions) produced bypartial acid hydrolysis (type A gelatin) or by partial alkalinehydrolysis (type B gelatin) of animal collagen, most commonly derivedfrom cattle, pig, and fish sources. Gelatin can be obtained in varyingmolecular weight ranges. Recombinant sources of gelatin may also beused.

As used herein, the term “polyethylene glycol (PEG)” refers to anoligomer or polymer of ethylene oxide. PEG is also known as polyethyleneoxide (PEO) or polyoxyethylene (POE). The structure of PEG is commonlyexpressed as H—(O—CH₂—CH₂)_(n)—OH.

As used herein, the term “immunogen” refers to any substance (e.g., anantigen, combination of antigens, pathogen fragment, whole pathogen)capable of eliciting an immune response in an organism. An “immunogen”is capable of inducing an immunological response against itself afteradministration to a mammalian subject. The term “immunological” as usedherein with respect to an immunological response, refers to thedevelopment of a humoral (antibody mediated) and/or a cellular (mediatedby antigen-specific T cells or their secretion products) responsedirected against an immunogen in a recipient subject. Such a responsecan be an active response induced by administration of an immunogen orimmunogenic peptide to a subject or a passive response induced byadministration of antibody or primed T cells that are directed towardsthe immunogen. In some embodiments, an immunogen is an influenza virus.In some embodiments, an immunogen is a viral vaccine (e.g., a monovalent(also called univalent) or a multivalent (also called polyvalent)vaccine, such as for influenza). In some embodiments, the vaccine (e.g.,influenza vaccine may be tetravalent or quadrivalent). In someembodiments, the immunogen is a replicating or non-replicating vaccinevector (e.g., comprises an adenovirus vector, an adeno-associated virusvector, an alpha virus vector, a herpesvirus vector, a measles virusvector, a poxvirus vector, or a vesicular stomatitis virus vector). Insome embodiments, the immunogen is an enterovirus, a flavivirus, arotavirus, a measles virus, a mumps virus, a rubella virus, or afragment thereof. In some embodiments, an inactivated or live attenuatedpolio virus, or antigenic fragment thereof, is an immunogen. In someembodiments, an inactivated or live attenuated rotavirus, or antigenicfragment thereof, is an immunogen. In some embodiments, an inactivated,live attenuated or recombinant flavivirus, or antigenic fragmentthereof, is an immunogen.

As used herein, the term “immunogenicity” refers to the ability of asubstance, such as an antigen or epitope, to provoke humoral and/orcell-mediated immunological response in a subject. A skilled artisan canreadily measure immunogenicity of a substance. The presence of acell-mediated immunological response can be determined by anyart-recognized methods, e.g., proliferation assays (CD4+ T cells), CTL(cytotoxic T lymphocyte) assays, or immunohistochemistry with tissuesection of a subject to determine the presence of activated cells suchas monocytes and macrophages after the administration of an immunogen.One of skill in the art can readily determine the presence ofhumoral-mediated immunological response in a subject by anywell-established methods. For example, the level of antibodies producedin a biological sample such as blood can be measured by western blot,ELISA or other methods known for antibody detection.

As used interchangeably herein, the terms “implantable sustained-releasetip” or “releasable tip” refers to the distal end, e.g., tip, of amicroneedle capable of piercing a biological barrier, e.g., the skin,mucous surface, or buccal cavity, of a subject and being depositedwithin the biological barrier, a skin layer (e.g., the dermis). Inembodiments, the tip comprises a silk fibroin protein in an amountsufficient to sustain the release of a therapeutic agent, such as avaccine, antigen, and/or immunogen for a prolonged period of time, e.g.,for at least about 4 days (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or 14 or more days, e.g., between about 4 days and about 14days, e.g., between about 1-2 weeks, about 1-3 weeks, or about 1-4weeks). In some embodiments, the implantable sustained-release tipcomprises an influenza vaccine, antigen, and/or immunogen.

As used herein, the term “microneedle” refers to a structure having atleast two, more typically, three components, e.g., layers, for transportor delivery of a therapeutic agent, such as a vaccine, an antigen,and/or an immunogen, across a biological barrier, such as the skin,tissue, or cell membrane. In some embodiments, a microneedle comprises abase (e.g., a dissolvable base as described herein), a tip (e.g., animplantable tip as described herein), and optionally, a backingmaterial. In embodiments, a microneedle has dimension of between about350 μm to about 1500 μm in height (e.g., between about 350 μm to about1500 μm, e.g., about 350 μm, about 400 μm, about 450 μm, about 500 μm,about 550 μm, about 600 μm, about 650 μm, about 700 μm, about 750 μm,about 800 μm, about 850 μm, about 900 μm, about 950 μm, about 1000 μm,about 1050 μm, about 1100 μm, about 1150 μm, about 1200 μm, about 1250μm, about 1300 μm, about 1350 μm, about 1400 μm, about 1450 μm, about1500 μm)). In some embodiments, the microneedle is fabricated to haveany dimension and/or geometry to enable the deployment of an implantablesustained-release at a depth between about 100 μm and about 900 μm(e.g., at a depth of about 800 μm) into the dermis layer of the skin forcontrolled- or sustained-release of a vaccine.

As used herein, the term “microneedle patch” and “microneedle array”refers to a device comprising a plurality of microneedles, e.g., silkfibroin-based microneedles, e.g., arranged in a random or predefinedpattern, such as an array.

As used herein, the term “silk fibroin” includes silkworm fibroin andinsect or spider silk protein. Any type of silk fibroin can be usedaccording to various aspects described herein. Silk fibroin produced bysilkworms, such as Bombyx mori, is the most common and represents anearth-friendly, renewable resource. For instance, silk fibroin used in amicroneedle (e.g., an implantable controlled- or sustained-release tipof a microneedle) may be obtained by removing sericin from the cocoonsof B. mori. In some embodiments, the silk fibroin is a regenerated silkfibroin, e.g., a silk fibroin obtained after extraction of sericin fromthe cocoons of B. mori, and an additional processing e.g. via a boilingstep. Organic silkworm cocoons are also commercially available. Thereare many different silks, however, including spider silk (e.g., obtainedfrom Nephila clavipes), transgenic silks, recombinant and/or geneticallyengineered silks, such as silks from bacteria, yeast, mammalian cells,transgenic animals, or transgenic plants (see, e.g., WO 97/08315; U.S.Pat. No. 5,245,012), and variants thereof, that can be used.

As used herein, a “subject” refers to a human or animal. Usually theanimal is a vertebrate such as a primate, rodent, domestic animal orgame animal. Primates include chimpanzees, cynomolgus monkeys, spidermonkeys, and macaques (e.g., Rhesus). Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species (e.g.,domestic cat), canine species (e.g., dog, fox, wolf), avian species(e.g., chicken, emu, ostrich), and fish (e.g., trout, catfish andsalmon). In certain embodiments of the aspects described herein, thesubject is a mammal (e.g., a primate, e.g., a human). A subject can bemale or female. In certain embodiments, the subject is a mammal. Themammal can be a human, non-human primate, mouse, rat, dog, cat, horse,or cow, but are not limited to these examples. In addition, the methodsand formulations described herein can be used to treat domesticatedanimals and/or pets.

As used herein, the term “controlled- or sustained-release” refers tothe release of a therapeutic agent (e.g., from a microneedle,microneedle device, formulation, composition, article, device, andpreparation described herein, e.g., from a silk fibroin-basedmicroneedle tip as described herein), such as a vaccine, antigen, and/orimmunogen over a period of time, e.g., for at least about 1-14 days(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or moredays, e.g., between about 4 days and about 14 days, e.g., between about1-2 weeks, about 1-3 weeks, or about 1-4 weeks). In some embodiments,the controlled- or sustained-release of an vaccine, e.g., over a timeperiod of about 1 to about 14 days, e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, or 14 days, by a microneedle, microneedle device,formulation, composition, article, device, or preparation as describedherein can result, e.g., in broad-spectrum immunity in a subject. Insome embodiments, the vaccine formulations and preparations comprisingsilk fibroin have controlled- or sustained-release properties (e.g., areformulated and/or configured to release a vaccine, e.g., into the skinof the subject, over a period of, or at least 1, 5, 10, 15, 30, 45minutes; a period of, or at least, 1, 2, 3, 4, 5, 10, 24 hours; a periodof, or at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days; aperiod of, or at least, 1, 2, 3, 4, 5, 6, 7, 8 weeks; a period of, or atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months; a period of, or atleast, 1, 2, 3, 4, 5 years, or longer.

As used herein, the term “vaccine” refers to any preparation of anantigen (including subunit antigens, toxoid antigens, conjugateantigens, or other types of antigenic molecules) or a killed or liveattenuated microorganism that, when introduced into a subject's body,affects the immune response to the specific antigen or microorganism bycausing activation of the immune system against the specific antigen ormicroorganism (e.g., inducing antibody formation, T cell responses,and/or B-cell responses). Generally, vaccines against microorganisms aredirected toward at least part of a virus, bacteria, parasite,mycoplasma, or other infectious agent.

As used herein, the term “viruses” refers to an infectious agentcomposed of a nucleic acid encapsidated in a protein. Such infectiousagents are incapable of autonomous replication (i.e., replicationrequires the use of the host cell's machinery). Viral genomes can besingle-stranded (ss) or double-stranded (ds), RNA or DNA, and can orcannot use reverse transcriptase (RT). Additionally, ssRNA viruses canbe either sense (+) or antisense (−). Exemplary viruses include, but arenot limited to, dsDNA viruses (e.g., Adenoviruses, Herpesviruses,Poxviruses), ssDNA viruses (e.g., Parvoviruses), dsRNA viruses (e.g.,Reo viruses), (+)ssRNA viruses (e.g., Picomaviruses, Toga viruses),(−)ssRNA viruses (e.g., Orthomyxoviruses, Rhabdoviruses), ssRNA-RTviruses, i.e., (+)sense RNA with DNA intermediate in life-cycle (e.g.,Retroviruses), and dsDNA-RT viruses (e.g., Hepadnaviruses). In someembodiments, viruses can also include wild-type (natural) viruses,killed viruses, live attenuated viruses, modified viruses, recombinantviruses or any combinations thereof. Exemplary retroviruses includehuman immunodeficiency virus (HIV). Other examples of viruses include,but are not limited to, enveloped viruses, respiratory syncytialviruses, non-enveloped viruses (e.g., human papillomavirus (HPV)),bacteriophages, recombinant viruses, and viral vectors. The term“bacteriophages” as used herein refers to viruses that infect bacteria.

As used herein, the term “influenza virus” refers to a negative-sensessRNA virus within the Orthomyxoviridae family. An influenza virus canbe a live wild-type virus, a live attenuated virus, an inactivatedvirus, a chimeric virus, or a recombinant virus. Examples of influenzaviruses include influenza A, influenza B, and influenza C.

As used herein, the term “therapeutic agent” is art-recognized andrefers to any chemical moiety that is a biologically, physiologically,or pharmacologically active substance that acts locally or systemicallyin a subject. Examples of therapeutic agents, also referred to as“drugs”, are described in well-known literature references such as theMerck Index, the Physicians Desk Reference, and The PharmacologicalBasis of Therapeutics, and they include, without limitation,medicaments; vitamins; mineral supplements; substances used for thetreatment, prevention, diagnosis, cure or mitigation of a disease orillness, such as a viral infection; substances which affect thestructure or function of the body; or pro-drugs, which becomebiologically active or more active after they have been placed in aphysiological environment. Various forms of a therapeutic agent may beused which are capable of being released from the microneedles describedherein into adjacent tissues or fluids upon administration to a subject.Examples include steroids and esters of steroids (e.g., estrogen,progesterone, testosterone, androsterone, cholesterol, norethindrone,digoxigenin, cholic acid, deoxycholic acid, and chenodeoxycholic acid),boron-containing compounds (e.g., carborane), chemotherapeuticnucleotides, drugs (e.g., antibiotics, antivirals, antifungals),enediynes (e.g., calicheamicins, esperamicins, dynemicin,neocarzinostatin chromophore, and kedarcidin chromophore), heavy metalcomplexes (e.g., cisplatin), hormone antagonists (e.g., tamoxifen),non-specific (non-antibody) proteins (e.g., sugar oligomers),oligonucleotides (e.g., mRNA sequences or antisense oligonucleotidesthat bind to a target nucleic acid sequence), peptides, proteins,antibodies, photodynamic agents (e.g., rhodamine 123), radionuclides(e.g., I-131, Re-186, Re-188, Y-90, Bi-212, At-211, Sr-89, Ho-166,Sm-153, Cu-67 and Cu-64), toxins (e.g., ricin), and transcription-basedpharmaceuticals.

Microneedles Devices

The invention provides, silk fibroin-based microneedles and microneedledevices (e.g., microneedle arrays and patches) for the transport andrelease, e.g., controlled- or sustained-release, of a therapeutic agent,such as a vaccine, an antigen, and/or an immunogen (e.g., an influenzavaccine) across a biological barrier, such as the skin, a mucousmembrane, a buccal cavity, a tissue, or a cell membrane. Themicroneedles described herein can be in any shape and/or geometrysuitable for use in piercing a biological barrier, e.g., a layer of theskin, to enable release, e.g., controlled- or sustained-release, of avaccine within a subject. Non-limiting examples of the shape and/orgeometry of the microneedles include: a cylindrical shape, awedge-shape, a cone-shape, a pyramid-shape, and/or an irregular-shape,or any combinations thereof.

In some embodiments, a microneedle of the invention can comprise thefollowing layers: (1) a backing material; (2) a dissolvable base; and(3) an implantable controlled- or sustained-release tip. For example,the microneedles described herein may include a backing material appliedto a dissolvable base layer that supports a distal controlled- orsustained-release implantable tip comprising a silk fibroin and vaccine(e.g., an influenza vaccine, antigen, and/or immunogen).

In some embodiments, the length of the microneedle can be between about350 μm to about 1500 μm ((e.g., about 350 μm, about 400 μm, about 450μm, about 500 μm, about 550 μm, about 600 μm, about 650 μm, about 700μm, about 750 μm, about 800 μm, about 850 μm, about 900 μm, about 950μm, about 1000 μm, about 1050 μm, about 1100 μm, about 1150 μm, about1200 μm, about 1250 μm, about 1300 μm, about 1350 μm, about 1400 μm,about 1450 μm, about 1500 μm). In embodiments, the length ofmicroneedles can be fabricated sufficiently long enough to enabledelivery of an implantable tip comprising a vaccine, an antigen, and/oran immunogen for controlled- or sustained-release, as described herein,to the epidermis (e.g., about 10 μm to 120 μm below the skin surface),e.g., to induce an immune response. In some embodiments, the length ofmicroneedles can be fabricated sufficiently long enough to enabledelivery of an implantable tip comprising a vaccine, an antigen, and/oran immunogen for controlled- or sustained-release, as described herein,to the dermis (e.g., about 60 μm to about 2.1 mm below the skinsurface). An skilled artisan can adjust the microneedle length for anumber of factors, including, without limitations, tissue thickness,e.g., skin thickness, (e.g., as a function of age, gender, location onbody, species (e.g., animal), drug delivery profile, diffusionproperties of the vaccine, antigen, and/or immunogen for controlled- orsustained-release (e.g., the ionic charge and/or molecule weight, and/orshape of the vaccine, antigen, and/or immunogen for controlled- orsustained-release), or any combinations thereof. However, withoutwishing to be bound by theory, with an approximately 650 μm tallmicroneedle an implantable sustained-release tip may be deployed at adepth of between about 100 μm and about 600 μm within the dermis layerof the skin to a subject to achieve controlled- or sustained-release ofvaccine from the tip. In some embodiments, the microneedle may be about800 μm tall (e.g., between about 500 μm and 1200 μm tall).

Exemplary microneedles of the invention are depicted in FIGS. 5A-5B.

In some embodiments, a plurality of microneedles can be arranged in arandom or predefined pattern to form a microneedle array and/or patch,as described herein. The patch may comprise a carrier, backing, or“handle” layer adhered to the back of the base (see, e.g., FIG. 4). Thislayer can provide structural support and an area by which the patch canbe handled and manipulated without disturbing the needle array.

Microneedle Array

The microneedle array may comprise about 121 needles in an 11×11 squaregrid with approximately 0.75 mm pitch. Individual needles are conesapproximately 0.65 mm long with base diameter approximately 0.35 mm andincluded angle of approximately 30°. The tip of the needle must be sharpin order to penetrate the skin. The radius of curvature of the tipshould ideally be no more than 0.01 mm.

Backing

Exemplary backing materials that can be used in the fabrication of amicroneedle of the invention include, but are not limited a solidsupport, e.g., a paper-based material, a plastic material, a polymericmaterial, or a polyester-based material (e.g., a Whatman 903 paper, apolymeric tape, a plastic tape, an adhesive-backed polyester tape, orother medical tape). In some embodiments, the backing comprises aWhatman 903 paper. In some embodiments, the backing comprises apolyester tape. In some embodiments, the polyester tape comprises anadhesive-backed polyester tape. In some embodiments, the backingmaterial may be coated (e.g., at least on one side) with an adhesivesuitable for bonding to and/or adhering to the dissolvable base of amicroneedle described herein.

The backing materials used in the microneedles of the invention may havevarious properties, including, but not limited to, the ability to bondand/or adhere to the dissolving base layer to permit demolding. Abacking material must be strong enough for the backing to maintain patchintegrity, e.g., if the dissolving base layer has cracks ordiscontinuities. The backing material may be sufficiently flexible so asto conform, for example, to a non-flat surface, such as a skin surface.In particular, the backing must be flexible enough during wear time,such as after the patch is applied (e.g., pressed into) the skin. Thebacking may comprise and/or consist of a non-dissolving material, suchthat the backing maintains its integrity after patch application to askin surface and during patch removal from a skin surface.

The backing may have any dimension suitable for application to a targetskin surface. In some embodiments, the dimensions of the backing can bea 12 mm diameter circle. In some embodiments, the dimensions of thebacking can be a 12 mm wide strip with a “handle” section of up to 12 mmlength beyond the edge of the 12 mm×12 mm patch.

Dissolvable Base

The dissolving base layer forms the base of the conical needles (e.g.,functions as the support for the distal silk fibroin tips that areloaded with a vaccine, an antigen, and/or an immunogen). The dissolvablebase layer can also function as a layer connecting adjacent needles toform a microneedle array or patch. In some embodiments, the dissolvablebase layer comprises less than 98% (e.g., less than about 98%, less thanabout 90%, less than about 80%, less than about 70%, less than about60%, less than about 50%, less than about less 40%, less than about 30%,less than about 20%, less than about 10%, less than about 9%, less thanabout 8%, less than about 7%, less than about 6%, less than about 5%,less than about 4%, less than about 3%, less than about 2%, less thanabout 1%) of the total amount (e.g., dose) of a vaccine, an antigen,and/or an immunogen comprises loaded into the microneedle and/ormicroneedle device. In some embodiments, the dissolvable base layer doesnot comprise, e.g., a detectable amount of, a vaccine, an antigen,and/or an immunogen. In some embodiments, dissolvable base layer isformulated to limit and/or reduce the amount of vaccine, antigen, and/orimmunogen leakage (e.g., diffusion) from the silk fibroin tips into thedissolvable base layer, e.g., as compared to art known base layerformulations, e.g., base layer formulations comprising PAA. In someembodiments, a limit and/or reduce amount of vaccine, antigen, and/orimmunogen leakage (e.g., diffusion) from the silk fibroin tips can bedetermined about 1 day, about 2 days, about 3 days, about 4 days, about5 days, or about 6 days; about 1 week, about 2 weeks, or about 3 weeks;about 1 month, about 2 months, about 3 months, about 4 months, about 5months, about 6 months, about 7 months, about 8 months, about 9 months,about 10 months, or about 11 months; or about 1 year or more afterfabrication and storage (e.g., storage at about 4° C. (e.g.,refrigeration), at about 25° C. (e.g., room temperature), at about 37°C. (e.g., body temperature), at about 45° C. and/or at about 50° C.),e.g., as compared to a base layer formulation comprising PAA.

The dissolvable base layer comprises a material that can dissolve intothe skin, e.g., within the intended wear time (e.g., about fiveminutes). In some embodiments, the at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100% of the dissolvable base layer isdissolved after application, e.g., to the skin, within the intended weartime (e.g., about 1 minute, about 2 minutes, about 3 minutes, about 4minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8minutes, about 9 minutes, or about 10 minutes or more).

The material used in the fabrication of the dissolvable base must besufficiently strong enough to enable the microneedle to penetrate theskin, and be tough enough (e.g., not brittle) to also enable demolding.The dissolvable base material must be amenable to routine handlingwithout catastrophic failure, and must retain its mechanical propertiesbetween demolding and application (e.g., not so hygroscopic that itmelts due to ambient humidity). The dissolvable base layer material mustbe non-toxic and non-reactogenic at the doses used in a patch. In someembodiments, the dissolvable base layer comprises a water solublecomponent. In some embodiments, a dissolvable base layer, as describedherein, has improved biocompatibility, e.g., as compared to adissolvable base layer comprising poly(acrylic acid) (PAA). In someembodiments, the dissolvable base layer material causes a reducedinflammatory response and/or reduced tissue necrosis. In someembodiments, the dissolvable base layer material is not PAA, and inducesa reduced inflammatory response and/or reduced tissue necrosis comparedto PAA. In some embodiments, the dissolvable base layer material has apH similar to that of the biological barrier into which it will bedissolved, e.g., a pH of about 4.0 to about 8.0

Non-limiting examples of materials that may be used to fabricate thedissolvable base layer include gelatin (e.g., hydrolyzed gelatin),polyethylene glycol (PEG), sucrose, low-viscosity carboxymethylcellulose(CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronate,maltose, and/or methyl cellulose. In some embodiments, the dissolvablebase comprises one, two, three, four, five, six, seven, eight, or more(e.g., all) of gelatin, polyethylene glycol (PEG), sucrose,carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA), hyaluronate, maltose, and methyl cellulose, e.g., at aconcentration between about 1% and about 75% (e.g., about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, or about 75%). In some embodiments, the dissolvable base does notcomprise a therapeutic agent, as described herein.

In some embodiments, the dissolvable base comprises between about 10%and about 70% gelatin (e.g., hydrolyzed gelatin) (e.g., about 10%, about20%, about 30%, about 40%, about 50%, about 60%, or about 70% gelatin).

In some embodiments, the dissolvable base comprises between about 1% andabout 70% polyethylene glycol (PEG) (e.g., about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, or about 70%PEG).

In some embodiments, the dissolvable base comprises between about 1% andabout 35% sucrose (e.g., about 1%, about 2%, about 3%, about 4%, about5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about20%, about 25%, about 30%, or about 35% sucrose).

In some embodiments, the dissolvable base comprises between about 1% andabout 35% CMC (e.g., about 1%, about 2%, about 3%, about 4%, about 5%,about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%,about 25%, about 30%, or about 35% CMC).

In some embodiments, the dissolvable base comprises between about 10%and about 70% PVP (e.g., about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, or about 70% PVP).

In some embodiments, the dissolvable base comprises between about 1% andabout 35% PVA (e.g., e.g., about 1%, about 2%, about 3%, about 4%, about5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about20%, about 25%, about 30%, or about 35% PVA).

In some embodiments, the dissolvable base comprises between about 1% andabout 75% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about25%, about 30%, or about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, or about 75% hyaluronate). In someembodiments, the dissolvable base comprises between about 1% and about75% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about25%, about 30%, or about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, or about 75% maltose).

In some embodiments, the dissolvable base comprises between about 1% andabout 75% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about25%, about 30%, or about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, or about 75% methyl cellulose).

In some embodiments, the dissolvable base layer may comprise 40%hydrolyzed gelatin, 10% Sucrose w/v in DI water. Optionally, the baselayer may include 1% low-viscosity carboxymethylcellulose (CMC), whichmay reduce brittleness. In some embodiments, the dissolvable base layermay comprise polyvinylpyrrolidone (PVP) of 10 kD MW at up to 50% w/v inDI water; polyvinyl alcohol (PVA) 87% hydrolyzed at 13 kD MW at up to20% in DI water; or CMC at up to 10% in DI water. The followingcombinations may also be suitable for use in the fabrication of adissolvable base layer: 30% PVP and 10% PVA; 37% PVP, 5% PVA, and 15%sucrose; or various other proportions of PVP, PVA, and sucrose.

In some embodiments, the dissolvable base layer is approximately 12 mmsquare and 0.75 mm thick. In some embodiments, the dissolvable baselayer can cover the entire patch. In some embodiments, the dimension ofthe base layer can be a 12 mm diameter circle, or a 12×12 mm square.

Implantable Sustained-Release Tip

In embodiments, the implantable sustained-release tip can be fabricatedfrom silk fibroin and may comprise a vaccine, an antigen, and/or animmunogen as described herein (e.g., an influenza vaccine). In someembodiments, the implantable sustained-release tip can be designed to bedeployed into the dermis layer of the skin (e.g., not into thesubcutaneous space), as the population of professional antigenpresenting cells in the dermis is much higher than in the subcutaneousspace. In humans, the dermis ranges from about 1000-2000 μm (e.g., about1-2 mm) thick based on location and patient age and health. In rodents,the dermis is much thinner (e.g., mice ˜100-300 μm, and rats ˜800-1200μm). Without wishing to be bound by theory, with a 650 μm tallmicroneedle an implantable sustained-release tip may be deployed at adepth of between about 100 μm and about 600 μm to achieve thecontrolled- or sustained-release of a vaccine, an antigen, and/or animmunogen as described herein (e.g., an influenza vaccine).

Without being bound by theory, the molecular weight of the silk fibroinsolution used in the fabrication of a microneedle described herein canfunction as a control factor to modulate the controlled- orsustained-release of a vaccine, an antigen, and/or an immunogen (e.g.,an influenza vaccine) from the tip. In some embodiments, a highermolecular weight silk fibroin solutions can favor a slower controlled-or sustained-release (e.g., reducing the amount of an initial burst(e.g., the amount released on Day 0) by at least about 10% and thenreleasing additional antigen over at least about the next 4 days). Insome embodiments, the controlled- or sustained-release of a vaccine, anantigen, and/or an immunogen (e.g., an influenza vaccine) from the tipmay be over at least about 4 days (e.g., about 4, 5, 6, 7, 8, 9, 10, 11,12, 13, or 14 or more days, e.g., between about 4 days and about 14days, e.g., between about 1-2 weeks, about 1-3 weeks, or about 1-4weeks). In some embodiments, controlled- or sustained-release occursover about 1 week to about 2 weeks.

In embodiments, the silk fibroin solution used in the fabrication of amicroneedle described herein can be a low molecular weight silk fibroincomposition comprising a population of silk fibroin fragments having arange of molecular weights, characterized in that: no more than 15% ofthe total number of silk fibroin fragments in the population has amolecular weight exceeding 200 kDa, and at least 50% of the total numberof the silk fibroin fragments in the population has a molecular weightwithin a specified range, wherein the specified range is between about3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa.Stated another way, the silk fibroin solution used in the fabrication ofa microneedle described herein can comprise a population of silk fibroinfragments having a range of molecular weights, characterized in that: nomore than 15% of the total moles of silk fibroin fragments in thepopulation has a molecular weight exceeding 200 kDa, and at least 50% ofthe total moles of the silk fibroin fragments in the population has amolecular weight within a specified range, wherein the specified rangeis between about 3.5 kDa and about 120 kDa, or between about 5 kDa andabout 125 kDa. (see, e.g., WO2014/145002, incorporated herein byreference herein).

Exemplary silk fibroin (e.g., regenerated silk fibroin) solutions mayhave different molecular weight profiles are shown as determined by sizeexclusion chromatography (SEC) methods (see, e.g., FIG. 5). In someembodiments, the silk fibroin solutions can be prepared, e.g., accordingto established methods. In some embodiments, pieces of cocoons from thesilkworm Bombyx mori were first boiled in 0.02 M Na2CO3 to removesericin protein which is present in unprocessed, natural silk, prior toanalysis by SEC. In some embodiments, silk fibroin composition can be acomposition or mixture produced by degumming cocoons from the silkwormBombyx mori at an atmospheric boiling temperature for about 480 minutesor less, e.g., less than 480 minutes, less than 400 minutes, less than300 minutes, less than 200 minutes, less than 180 minutes, less than 120minutes, less than 100 minutes, less than 60 minutes, less than 50minutes, less than 40 minutes, less than 30 minutes, less than 20minutes, less than 10 minutes or shorter. In one embodiment, the silkfibroin composition can be a composition or mixture produced bydegumming silk cocoon at an atmospheric boiling temperature in anaqueous sodium carbonate solution for about 480 minutes or less, e.g.,less than 480 minutes, less than 400 minutes, less than 300 minutes,less than 200 minutes, less than 180 minutes, less than 120 minutes,less than 100 minutes, less than 60 minutes, less than 50 minutes, lessthan 40 minutes, less than 30 minutes, less than 20 minutes, less than10 minutes or shorter.

In some embodiments, the silk fibroin solution may be a 10-minute boil(10 MB), a 60-minute boil (60 MB), a 120-minute boil (120 MB), a180-minute boil (180 MB), or a 480-minute boil (480 MB) silk fibroinsolution (see, e.g., FIG. 5). In some embodiments, an influenza vaccine,antigen, and/or immunogen can be formulated in a 1% w/v to about 10% w/v(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w/v) 10 MB silk fibroinsolution. In some embodiments, an influenza vaccine, antigen, and/orimmunogen can be formulated in a 1% w/v to about 10% w/v (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, or 10% w/v) 60 MB silk fibroin solution. In someembodiments, an influenza vaccine, antigen, and/or immunogen can beformulated in a 1% w/v to about 10% w/v (e.g., about 1, 2, 3, 4, 5, 6,7, 8, 9, or 10% w/v) 120 MB silk fibroin solution. In some embodiments,an influenza vaccine, antigen, and/or immunogen can be formulated in a1% w/v to about 10% w/v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%w/v) 180 MB silk fibroin solution. In some embodiments, an influenzavaccine, antigen, and/or immunogen can be formulated in a 1% w/v toabout 10% w/v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w/v) 480 MBsilk fibroin solution.

Without being bound by theory, the primary tunability of the implantablesustained-release tip is its crystallinity, measured via beta-sheetcontent (intermolecular and intramolecular β-sheet). This impacts thesolubility of the silk tip matrix and the ability of antigen to beretained. With the increased β-sheet content, the tip also becomes moremechanically strong. Specific vaccine release profiles are achievedthrough modulation of the crystallinity and the diffusivity of the silkmatrix. This is accomplished through both silk input material andformulation as well as post-treatment to increase crystallinity (e.g.water annealing, methanol/solvent annealing). In some embodiments, theimplantable controlled- or sustained-release microneedle tip comprises abeta-sheet content of between about 10% and about 60% (e.g., about 10%,about 20%, about 30%, about 40%, about 50%, about 60%), e.g., as basedon a “crystallinity index,” e.g., a “crystallinity index” known in theart. In some embodiments, the implantable controlled- orsustained-release microneedle tip can be formulated as a particle (e.g.,a microparticle and/or a nanoparticle).

Dimensions of the Implantable Sustained-Release Tip

The methods provided herein can be used to fabricate silk fibroin-basedimplantable sustained-release tips of any dimensions, e.g., ranging fromabout 75 μm to about 800 μm in height/length (e.g., about 75, about 100μm, about 125 μm, about 150 μm, about 250 μm to about 300 μm, about 300μm to about 350 μm, about 350 μm to about 400 μm, about 400 μm to about450 μm, about 450 μm to about 500 μm, about 500 μm to about 550 μm,about 550 μm to about 600 μm, about 600 μm to about 650 μm, about 650 μmto about 700 μm, about 700 μm to about 750 μm, about 750 μm, to about800 μm), and/or having a tip radius of about 10 μm or less (e.g.,between about 1 μm and about 10 μm, e.g., about 1 μm or less, about 2 μmor less, about 3 μm or less, about 4 μm or less, about 5 μm or less,about 6 μm or less, about 7 μm or less, about 8 μm or less, about 9 μmor less, or about 10 μm or less). In some embodiments, the implantabletip can have a diameter of any size, e.g., based upon the type ofbiological barrier (e.g., skin layer) intended to be pierced by the tip.In embodiments, the tip can have a dimension (e.g., a diameter) rangingfrom about 50 nm to about 50 μm (e.g., about 50 nm to about 250 nm,about 250 nm to about 500 nm, about 500 to about 750 nm, about 750 nm toabout 1 μm, about 1 μm to about 5 μm, about 5 μm to about 10 μm, about10 μm to about 15 μm, about 15 μm to about 20 μm, about 20 μm to about25 μm, about 25 μm to about 30 μm, about 30 μm to about 35 μm, about 35μm to about 40 μm, about 40 μm to about 45 μm, or about 45 μm to about50 μm). It can be understood that there is no fundamental limitationpreventing the sustained-release tips from having even smaller diameters(e.g., the limit of silk replica casting has been demonstrated with aresolution of tens of nm, see, e.g., Perry et al., 20 Adv. Mat. 3070(2008)).

In some embodiments, the sharpness of the implantable sustained-releasetip point is described herein in terms of tip radius. The molds used inthe fabrication of the microneedles described herein are designed tohave a tip radius between about 0.5 μm to about 10 μm (e.g., about 0.5μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm,7 μm, 8 μm, 9 μm, or 10 μm). In some embodiments, the tip radius isbetween about 20 μm to about 25 μm (e.g., about 20 μm, 21 μm, 22 μm, 23μm, 24 μm, or 25 μm). Without being bound by theory, it can beunderstood that blunter needles may require more force to penetrate theepidermis. In embodiments, other dimensions of the implantablesustained-release tip may be controlled by the shape of the mold andfill volume. In some embodiments, the implantable sustained-release tiphave an included angle between about 5 degrees and about 45 degrees(e.g., about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 degrees).In some embodiments, the implantable sustained-release tip can have anincluded angle between about 15 degrees and 45 degrees (e.g., about 15degrees, about 16 degrees, about 17 degrees, about 18 degrees, about 19degrees, about 20 degrees, about 21 degrees, about 22 degrees, about 23degrees, about 24 degrees, about 25 degrees, about 26 degrees, about 27degrees, about 28 degrees, about 29 degrees, about 30 degrees, about 31degrees, about 32 degrees, about 33 degrees, about 34 degrees, about 35degrees, about 36 degrees, about 37 degrees, about 38 degrees, about 39degrees, about 40 degrees, about 41 degrees, about 42 degrees, about 43degrees, about 44 degrees, or about 45 degrees.

In embodiments, the height of the implantable sustained-release tip maydepend on the formulation and print volume, which can influence thesurface tension and drying kinetics. In some embodiments, the height ofthe implantable sustained-release tip may extend to half of the fullheight of the microneedle. In some embodiments, the height of theimplantable sustained-release tip is between about 75 μm to about 475 μm(e.g., about 75, about 100 μm, about 125 μm, about 150 μm, about 175 μm,about 200 μm, about 225 μm, about 250 μm, about 275 μm, about 300 μm,about 325 μm, about 375 μm, about 400 μm, about 425 μm, or about 475μm). In some embodiments, the base of the tip comprises a thin“shell”-like layer roughly between about 5-10 μm thick (e.g., about 5,6, 7, 8, 9, or 10 μm thick). In some embodiments, the implantablesustained-release tip may dry to a more solid construct with minimal“shell” wherein the height may be closer to 150 μm (e.g., between about50 μm and about 200 μm) and the thickness >50 μm (e.g., between about 25μm and about 75 μm).

Further, the microneedles of the present invention can take advantage ofart known techniques developed, e.g., to functionalize silk fibroin(e.g., active agents such as dyes and sensors). See, e.g., U.S. Pat. No.6,287,340, Bioengineered anterior cruciate ligament; WO 2004/000915,Silk Biomaterials & Methods of Use Thereof; WO 2004/001103, SilkBiomaterials & Methods of Use Thereof; WO 2004/062697, Silk FibroinMaterials & Use Thereof; WO 2005/000483, Method for Forming inorganicCoatings; WO 2005/012606, Concentrated Aqueous Silk Fibroin Solution &Use Thereof; WO 20111005381, Vortex-Induced Silk fibroin Gelation forEncapsulation & Delivery; WO 20051123114, Silk-Based Drug DeliverySystem; WO 2006/076711, Fibrous Protein Fusions & Uses Thereof in theFormation of Advanced Organic/Inorganic Composite Materials; U.S.Application Pub. No. 2007/0212730, Covalently immobilized proteingradients in three-dimensional porous scaffolds; WO 2006/042287, Methodfor Producing Biomaterial Scaffolds; WO 2007/016524, Method for StepwiseDeposition of Silk Fibroin Coatings; WO 2008/085904, BiodegradableElectronic Devices; WO 20081118133, Silk Microspheres for Encapsulation& Controlled Release; WO 20081108838, Microfluidic Devices & Methods forFabricating Same; WO 20081127404, Nanopatterned Biopolymer Device &Method of Manufacturing Same; WO 20081118211, Biopolymer PhotonicCrystals & Method of Manufacturing Same; WO 20081127402, BiopolymerSensor & Method of Manufacturing Same; WO 20081127403, BiopolymerOptofluidic Device & Method of Manufacturing the Same; WO 20081127401,Biopolymer Optical Wave Guide & Method of Manufacturing Same; WO20081140562, Biopolymer Sensor & Method of Manufacturing Same; WO20081127405, Microfluidic Device with Cylindrical Microchannel & Methodfor Fabricating Same; WO 20081106485, Tissue-Engineered Silk Organs; WO20081140562, Electroactive Bioploymer Optical & Electro-Optical Devices& Method of Manufacturing Same; WO 20081150861, Method for Silk FibroinGelation Using Sonication; WO 20071103442, Biocompatible Scaffolds &Adipose-Derived Stem Cells; WO 20091155397, Edible Holographic SilkProducts; WO 20091100280, 3-Dimensional Silk HydroxyapatiteCompositions; WO 2009/061823, Fabrication of Silk Fibroin PhotonicStructures by Nanocontact Imprinting; WO 20091126689, System & Methodfor Making Biomaterial Structures.

In various embodiments, the silk fibroin-based microneedle tips canfurther comprise at least one additional therapeutic agent, wherein theadditional therapeutic can be dispersed throughout the microneedle orform at least a portion of the microneedle tip. In some embodiments, theadditional therapeutic agent is useful in the treatment of a viralinfection described herein. Optionally the silk fibroin-basedmicroneedle tips can further comprise an excipient and/or adjuvant, asdescribed herein.

Methods of Making and/or Manufacturing a Microneedle

A schematic diagram and a flow chart depicting the method of fabricationof a microneedle of the invention are shown in FIGS. 3 and 4,respectively. Machine vision guided printing of precise nL volumes ofsilk fibroin solution into individual needle cavities enables differentdosages and formulations to be incorporated within releasable tips of amicroneedle device (e.g., a microneedle array or patch). An exemplarymicroneedle device (e.g., a microneedle array or patch), comprises an11×11 cone array. It should be understood that the microneedle devicemay include needle cavities produced in an array of varying number ofcavities and orientations to achieve a desired result.

Mold Production

In some embodiments, a mold is used in the fabrication of a microneedledevice. As will be discussed in greater detail below, a sterilized moldis used to produce a microneedle device having an array of releasabletips embodying an antigen-silk formulation.

For example, a silicone (DOW Corning Sylgard® 184) resin may be castagainst a positive master having the intended geometry of a microneedlearray. Once the silicone has cured, it may be removed from the master.The master can then be reused for a large number of silicone castings.Throughout the fabrication process the silicone mold may be inspectedfor defects (e.g., between castings). If desired, the silicone mold canbe sterilized, for example, by autoclaving. In one embodiment, the moldincludes a mold body having an array of needle cavities formed withinthe mold body.

In some embodiments, other types of silicone and/or other materials andprocesses may be used to fabricate the mold. For example, liquidsilicone injection molding and thermoplastic elastomer injection moldingmay be used. Without wishing to be bound by theory, it may be understoodthat a key requirement is that the mold material be soft and flexible(e.g., comprise a Shore hardness of about 50 A) and have low adhesionwith silk and other materials used in the construction of the patch.

Tip Filling

Tip formulation consisting of silk fibroin, antigen, and potentiallyother excipients in aqueous solution, is dispensed into each needlecavity in the mold via nanoliter printing. Currently this is done at labscale using a Biojet Elite™ AD3400 dispensing system produced by BioDot,but systems with similar capabilities made by other suppliers can beemployed. The working volume of the BioDot™ dispenser is enclosed and ismaintained at 60% relative humidity (RH) to slow drying of theformulation and avoid buildup of dry solids on the dispensing nozzle.

Molds are placed within a fixture that constrains their locations on theprocessing platform of the BioDot™ dispenser. The machine uses a camerato image each mold and a machine vision algorithm identifies the preciselocation and orientation of the array of needle cavities in each mold.This location is used to direct the subsequent dispensing steps. Thefilled molds are inspected using a stereomicroscope for filling defectssuch as misaligned dispenses or large bubbles in the liquid.

Primary Drying

The filled molds are set aside to dry within the machine enclosure forabout 7 minutes. After drying, the above dispensing process is repeatedand the molds are dried again for 7 minutes. This is the “primary”drying step.

Secondary Drying

The molds are moved to a chamber in which humidity is controlled toabout 25% RH and ambient room temperature and kept overnight (about 14hours) to complete drying. This is the “secondary” drying step.

Water Annealing

The molds are transferred to a vacuum desiccator that also containsabout 500 mL of DIW. The desiccator is closed and vacuum is drawn forabout 5 minutes using the main vacuum line in the lab. After 5 minutes,the outlet valve of the desiccator is closed and it is placed within anincubator holding at 37° C. for four hours. After four hours, thedesiccator is vented and the molds are transferred back to the 25% RHchamber at ambient room temperature.

Post-Anneal Drying

Molds are kept at 25% RH for at least four hours or up to overnightbefore subsequent steps.

Base Layer Filling

The dissolvable base layer is formed by filling the mold with a solutionof 40% w/v Hydrolyzed Gelatin and 10% w/v Sucrose in DIW and then dryingthis layer. First, 150 μL of base solution is spread evenly over themold with a pipette. Next, the molds are centrifuged at 3900 rpm for 2minutes. The molds are inspected and if any needle cavities remainunfilled, the filling and centrifuging process is repeated. The moldsare “topped off” with 50 μL of base solution.

Base Drying

The filled molds are transferred back to the chamber at 25% RH and driedat least overnight and up to 3 days.

Backing Application

The patches used to generate the release, e.g., controlled- orsustained-release, and improved immunogenicity (see, e.g., the Examples)had a paper backing layer; however, subsequent development has shownthat adhesive plastic tape has superior performance as a backing layer.

The paper backing process is as follows: the dried base layer ispartially re-wetted with 10-30 μL of DIW spread over the surface with apipette. Whatman 903 paper is punched into 12 mm diameter circles. Thecircles of paper are gently pressed into the wet surface of the baselayer. The wet base layer partially soaks into the paper. The molds withbacking are transferred back into the 25% RH chamber to dry for at least4 hours until use.

Adhesive Tape Process

Adhesive-backed polyester tape (e.g., 3M® Magic™ tape) is cut into apiece about 12 mm wide and about 25 mm long. One end of the tape isaligned with the patch and gently pressed onto the surface of the baselayer. The free end of the tape is folder over onto itself to form anon-adhesive “handle.”

Demolding

The patches are removed from the mold before use. The flexible mold isgently bent away from the stiffer patch, and the patch is taken awayfrom the mold. The patch is inspected for defects such as missing orbroken needles.

Packaging

In the studies above, the patches were used soon after demolding andwere not packaged. If extended storage is needed, assembled patches canbe packaged in a container with low moisture vapor transmission rate(e.g., glass vial or thermoformed plastic tray made of low MVTRmaterials and a foil-backed heat-sealed lid) along with a desiccant tomaintain about rate between about 0% and about 50% (e.g., between about0% and 10%, between about 10% and about 20%, between about 20% and about30%, between about 30% and about 40%, or between about 40% and 50%,e.g., about 25%) relative humidity inside the package (see, e.g., FIG.7).

Viruses, Antigens, and Immunogens

The present invention provides, in some embodiments, the delivery, e.g.,the controlled- or sustained-delivery, of various therapeutic agents,such as vaccines, antigens, and/or immunogens derived from a virus thatis a member of the family Orthomyxovirus, e.g., by a formulation,composition, articles, device, preparations, microneedle and/ormicroneedle device (e.g., a microneedle patch) described herein and/oraccording to a method described herein. In some embodiments, a vaccine,a microneedle, and/or a microneedle device (e.g., a microneedle patch)described herein may comprise a negative-sense ssRNA virus and/or an RNAvirus, such as an influenza virus. In some embodiments, the vaccine,antigen, and/or immunogen comprises a nucleic acid (e.g., a DNA and/orRNA) derived from an influenza virus. In some embodiments, the vaccine,antigen, and/or immunogen comprises an amino acid (e.g., a peptideand/or protein) derived from an influenza virus. In some embodiments,the influenza vaccine, antigen, and/or immunogen comprise an inactivatedand/or a live attenuated virion, or split virion, of an influenza virus.In some embodiments, the vaccine and/or the microneedle comprises anon-replicating viral antigen.

In particular, the invention contemplates a vaccine, a microneedle,and/or a microneedle device (e.g., a microneedle patch) comprising aninfluenza virus vaccine, antigen, and/or immunogen. The influenza virusis a RNA virus (e.g., a linear negative-sense single stranded RNAvirus). There are four known genera of influenza virus, each containinga single type (e.g., Influenza A, B, C, and D). Influenza viruses cancontinuously change and are subject to both antigenic drift andantigenic shift. Exemplary influenza strains are further described inthe Examples (see, e.g., Tables 1 and 2).

Influenza A can be divided into subtypes on the basis of two proteins onthe surface of the virus: hemagglutinin (HA) and neuraminidase (NA).Influenza A comprises 18 known HA subtypes, referred to herein asH1-H18, and 11 known NA subtypes, referred to herein as N1-N11. Manydifferent combinations of HA and NA proteins may be found on the surfaceof the influenza A virus. For example, an “H1N1 virus” designates aninfluenza A virus subtype comprising an H1 protein and an N1 protein.Exemplary influenza A virus subtypes confirmed to infect humans include,but are not limited to, H1N1, H3N2, H2N2, H5N1, H7N7, H1N2, H9N2, H7N2,H7N3, H10N7, and H7N9. The H1N1 virus and H3N2 virus are currently ingeneral circulation among humans.

Exemplary Influenza B viruses may belong to, e.g., the B/Yamagatalineage and/or the B/Victoria lineage.

Vaccines

Non-limiting examples of influenza vaccines for use in the microneedlesand microneedle devices (e.g., microneedle patches) described herein caninclude a commercial vaccine, such as a seasonal vaccine, a pandemicvaccine, and/or a universal vaccine; egg-based vaccines, cell-culturebased vaccines; recombinant vaccines; live attenuated, inactivated wholevirus, split virion, and/or protein subunit vaccines; and adjuvantedvaccines. Various commercial influenza vaccines are listed below.Additionally, influenza vaccines comprising an mRNA, a DNA, a viralvector, and/or a virus-like particle (VLP) are suitable for use in themicroneedles and microneedle devices (e.g., microneedle patches)described herein. In some embodiments, the influenza vaccine may targetmatrix protein 1, matrix protein 2 (M2e), and/or nucleoprotein (NP) ofan influenza virus.

Vaccine Manufacturer Seasonal Influenza Vaccines Fluzone High DoseSanofi Pasteur Fluzone Quadrivalent Sanofi Pasteur Fluzone IntradermalQuadrivalent Sanofi Pasteur Afluria/Fluvax Seqirus Agriflu Seqirus FluadSeqirus Flucelvax Seqirus Fluvirin Seqirus Aggripal Seqirus FluMistQuadrivalent MedImmune Flublok Protein Sciences (Sanofi Pasteur)FluLaval GlaxoSmithKline Fluarix GlaxoSmithKline Influvac MylanPreflucel Nanotherapeutics Anflu Sinovac Biotech Pandemic InfluenzaVaccines Influenza Virus Vaccine, H5N1 Sanofi Pasteur PandemrixGlaxoSmithKline Panflu Sinovac Biotech Panflu 1 Sinovac Biotech

Vaccine Formulations and Composition for Controlled- orSustained-Release

At least one vaccine, antigen, and/or immunogen described herein (e.g.,at least one vaccine, antigen, and/or immunogen derived from aninfluenza virus described herein) can be incorporated into a variety offormulations, compositions, articles, devices, and/or preparations foradministration, e.g., to achieve controlled- and/or sustained release.More particularly, at least one vaccine, antigen, and/or immunogendescribed herein (e.g., at least one vaccine, antigen, and/or immunogenderived from an influenza virus described herein) can be formulated intoformulations, compositions, articles, devices, and/or preparations bycombination with appropriate, pharmaceutically acceptable carriers ordiluents, and can be formulated into preparations in semi-solid, solid,or liquid formats. In some embodiments, the formulations, compositions,articles, devices, and/or preparations described herein comprise silkfibroin. Exemplary formulations, compositions, articles, devices, and/orpreparations comprise: a microneedle (e.g., a microneedle device, e.g.,a microneedle patch, e.g., as described herein), an implantable device(e.g., a pump, e.g., a subcutaneous pump), an injectable formulation, adepot, a gel (e.g., a hydrogel), an implant, and a particle (e.g., amicroparticle and/or a nanoparticle). As such, administration of thecompositions can be achieved in various ways, including intradermal,intramuscular, transdermal, subcutaneous, or intravenous administration.Moreover, the formulations, compositions, articles, devices, and/orpreparations can be formulated and/or administered to achievecontrolled- and/or sustained release of the at least one vaccine,antigen, and/or immunogen described herein (e.g., at least one vaccine,antigen, and/or immunogen derived from an influenza virus describedherein).

In some embodiment, the vaccine (e.g., the influenza vaccine) isadministered, e.g., substantially sustained, over a period of, or atleast 1, 5, 10, 15, 30, 45 minutes; a period of, or at least, 1, 2, 3,4, 5, 10, 24 hours; a period of, or at least, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 days; a period of, or at least, 1, 2, 3, 4, 5, 6, 7,8 weeks; a period of, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11months; a period of, or at least, 1, 2, 3, 4, 5 years, or longer. In oneembodiment, the vaccine (e.g., the influenza vaccine) is administered asa controlled- or sustained release formulation, dosage form, or device.In certain embodiments, the vaccine (e.g., the influenza vaccine) isformulated for continuous delivery, e.g., intradermal, intramuscular,and/or intravenous continuous delivery. In some embodiments, thecomposition or device for the controlled- or sustained-release of thevaccine is chosen from: a microneedle (e.g., a microneedle device, e.g.,a microneedle patch), an implantable device (e.g., a pump, e.g., asubcutaneous pump), an injectable formulation, a depot, a gel (e.g., ahydrogel), an implant, or a particle (e.g., a microparticle and/or ananoparticle). In one embodiment, the vaccine (e.g., the influenzavaccine) is in a silk-based controlled- or extended release dosage formor formulation (e.g., a microneedle described herein). In oneembodiment, the vaccine (e.g., the influenza vaccine) is administeredvia an implantable device, e.g., a pump (e.g., a subcutaneous pump), animplant, an implantable tip of a microneedle, or a depot. The deliverymethod can be optimized such that a vaccine (e.g., an influenza vaccine)dose as described herein (e.g., a standard dose) is administered and/ormaintained in the subject for a pre-determined period (e.g., a periodof, or at least: 1, 5, 10, 15, 30, 45 minutes; 1, 2, 3, 4, 5, 10, 24hours 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days; 1, 2, 3, 4, 5,6, 7, 8 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months; 1, 2, 3, 4, 5years, or longer). The substantially sustained or extended release ofthe vaccine (e.g., the influenza vaccine) can be used for prevention ortreatment of a viral infection (e.g., an influenza viral infection) fora period of hours, days, weeks, months, or years.

The present invention provides, in some embodiments, formulations,compositions, articles, devices, and/or preparations of the inventioncan be formulated and/or configured for controlled- or sustained-releaseof a at least one vaccine, antigen, and/or immunogen (e.g., at least onevaccine, antigen, and/or immunogen derived from an influenza virusdescribed herein) in an amount (e.g., a dosage) and/or over a timeperiod sufficient to result in an immune response (e.g., a cellularimmune response and/or a humoral immune response) to the virus, e.g.,the influenza virus, in the subject.

In some embodiments, the formulations, compositions, articles, devices,and/or preparations of the invention can be formulated and/or configuredfor controlled- or sustained-release of a at least one vaccine, antigen,and/or immunogen (e.g., at least one vaccine, antigen, and/or immunogenderived from an influenza virus described herein) in an amount (e.g., adosage) and/or over a time period sufficient to result in broad spectrumimmunity in the subject.

The substantially continuously or extended release delivery orformulation of the vaccine (e.g., the influenza vaccine) can be used forprevention or treatment of a viral infection (e.g., an influenza viralinfection) for a period of hours, days, weeks, months, or years.

In some embodiments, at least one vaccine, antigen, and/or immunogendescribed herein can be added to the silk fibroin solution, e.g., beforeforming the silk fibroin microneedles or microneedle devices describedherein. In embodiments, a silk fibroin solution can be mixed with avaccine, antigen, and/or immunogen, and then used in the fabrication ofan implantable microneedle tip, e.g., by the process of filling and/orcasting, drying, and/or annealing to produce a microneedle having any ofthe desired material properties, as described herein.

Without being bound by theory, the ratio of silk fibroin to vaccine,antigen, and/or immunogen in an implantable tip of a microneedleinfluences their release. In some embodiments, increased silkconcentration in the implantable tip favors a slower release and/orgreater antigen retention within the tip. Any concentration of silk maybe used, as long as the concentration allows for printing and has themechanical strength sufficient to pierce the skin.

In some embodiments, silk fibroin can be used at a concentration rangingfrom about 1% w/v to about 10% w/v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, or 10% w/v) in the fabrication of a microneedle, or a componentthereof, as described herein.

Exemplary Excipients

In addition, the formulations, compositions, articles, devices, and/orpreparations can be formulated with common excipients, diluents orcarriers for administered by the intradermal, intramuscular,transdermal, subcutaneous, or intravenous routes. In some embodiments,the formulations, compositions, articles, devices, and/or preparationscan be administered, e.g., transdermally, and can be formulated ascontrolled- or sustained-release dosage forms and the like. Theformulations, compositions, articles, devices, and/or preparationsdescribed herein can be administered alone, in combination with eachother, or they can be used in combination with other known therapeuticagents.

Suitable formulations for use in the present invention are found inRemington's Pharmaceutical Sciences (1985). Moreover, for a review ofmethods for drug delivery, see, Langer (1990) Science 249:1527-1533. Theformulations, compositions, articles, devices, and/or preparationsdescribed herein can be manufactured in a manner that is known to thoseof skill in the art, e.g., by mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes. The following methods and excipients are merelyexemplary and are in no way limiting.

The silk fibroin formulations used in the fabrication of themicroneedles described herein may include excipients. In embodiments,inclusion of an excipient may be for the purposes of improving thestability of an incorporated vaccine, antigen, and/or immunogen; toincrease silk matrix porosity and diffusivity of the vaccine, antigen,and/or immunogen from the formulation, composition, article, device,preparation, and/or microneedle, e.g., microneedle tip; and/or toincrease crystallinity/beta-sheet content of silk matrix to render thesilk-material insoluble.

Exemplary excipients include, but are not limited to, a sugar or a sugaralcohol (e.g., sucrose, trehalose, sorbitol, mannitol, or a combinationthereof), a divalent cation (e.g., Ca²⁺, Mg²⁺, Mn²⁺, and Cu²⁺), and/orbuffers. In some embodiments, the concentration of an excipient can beused to modify the porosity of the matrix, e.g., with sucrose being usedas the most common excipient for this purpose. Excipients may also beadded to favor silk self-assembly into order beta-sheet secondarystructure, and such excipients generally can participate in hydrogenbonding or charge interactions with silk to achieve this effect.Non-limiting examples of excipients that can be used to favor silkself-assembly into order beta-sheet secondary structure includemonosodium glutamate (e.g., L-glutamic acid), lysine, sugar alcohols(e.g., sorbitol and/or glycerol), and solvents (e.g., DMSO, methanol,and/or ethanol).

In some embodiments, the sugar or the sugar alcohol is sucrose presentin an amount less than 70% (w/v), less than 60% (w/v), less than 50%(w/v), less than 40% (w/v), less than 30% (w/v), less than 20% (w/v),less than 10% (w/v), less than 9% (w/v), less than 8% (w/v), less than7% (w/v), less than 6% (w/v), or 5% (w/v) or less, e.g., immediatelybefore drying.

In some embodiments, the sugar or the sugar alcohol is sucrose presentin an amount between about 1% (w/v) to about 10% (w/v), about 2% (w/v)to about 8% (w/v), about 2.2% (w/v) to about 6% (w/v), about 2.4% (w/v)to about 5.5% (w/v), about 2.5 to about 5%, or about 2.4% (w/v), about2.5%, or about 5% (w/v), e.g., immediately before drying.

In some embodiments, the sugar or the sugar alcohol is trehalose presentin an amount between about 1% (w/v) to about 10% (w/v), about 2% (w/v)to about 8% (w/v), about 2.2% (w/v) to about 6% (w/v), about 2.4% (w/v)to about 5.5% (w/v), about 2.5 to about 5%, or about 2.4% (w/v), about2.5%, or about 5% (w/v), e.g., immediately before drying.

In some embodiments, the sugar or the sugar alcohol is sorbitol presentin an amount between about 1% (w/v) to about 10% (w/v), about 2% (w/v)to about 8% (w/v), about 2.2% (w/v) to about 6% (w/v), about 2.4% (w/v)to about 5.5% (w/v), about 2.5 to about 5%, or about 2.4% (w/v), about2.5%, or about 5% (w/v), e.g., immediately before drying.

In some embodiments, the sugar or the sugar alcohol is glycerol presentin an amount between about 1% (w/v) to about 10% (w/v), about 2% (w/v)to about 8% (w/v), about 2.2% (w/v) to about 6% (w/v), about 2.4% (w/v)to about 5.5% (w/v), about 2.5 to about 5%, or about 2.4% (w/v), about2.5%, or about 5% (w/v), e.g., immediately before drying.

In some embodiments, the vaccine preparation further comprising adivalent cation. In some embodiments, the divalent cation is selectedfrom the group consisting of Ca²⁺, Mg²⁺, Mn²⁺, and Cu²⁺. In someembodiments, the divalent cation is present in the preparation, e.g.,immediately before drying, in an amount between 0.1 mM and 100 mM. Insome embodiments, the divalent cation is present in the preparation,e.g., immediately before drying, in an amount between 10⁻⁷ and 10⁻⁴moles per standard dose of viral immunogen. In some embodiments, thedivalent cation is present in the preparation immediately before dryingin an amount between 10⁻¹⁰ to 2×10⁻³ moles.

In some embodiments, the vaccine preparation further comprisespoly(lactic-co-glycolic acid) (PGLA).

In some embodiments, the viral vaccine preparation further comprising abuffer, e.g., immediately before drying. In some embodiments, the bufferhas buffering capacity between pH 3 and pH 8, between pH 4 and pH 7.5,or between pH 5 and pH 7. In some embodiments, the buffer is selectedfrom the group consisting of HEPES and a CP buffer. In some embodiments,the buffer is present in the preparation, e.g., immediately beforedrying, in an amount between 0.1 mM and 100 mM. In some embodiments, thebuffer is present in an amount between 10⁻⁷ and 10⁻⁴ moles per standarddose of viral immunogen. In some embodiments, the buffer is present inan amount between 10⁻¹⁰ to 2×10⁻³ moles.

In addition, the vaccine can also be formulated as a depot, gel, orhydrogel preparation. Such long acting formulations can be administeredby implantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the vaccine can beformulated with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

In one embodiment, the vaccine is administered via an implantableinfusion device, e.g., a pump (e.g., a subcutaneous pump), an implant ora depot. Implantable infusion devices typically include a housingcontaining a liquid reservoir which can be filled transcutaneously by ahypodermic needle penetrating a fill port septum. The medicationreservoir is generally coupled via an internal flow path to a deviceoutlet port for delivering the liquid through a catheter to a patientbody site. Typical infusion devices also include a controller and afluid transfer mechanism, such as a pump or a valve, for moving theliquid from the reservoir through the internal flow path to the device'soutlet port.

In some embodiments, the vaccine can be packages and/or formulated as aparticle, e.g., a microparticle and/or a nanoparticle. Typicallynanoparticles are from 10, 15, 20, 25, 30, 35, 45, 50, 75, 100, 150 or200 nm or 200-1,000, e.g., 10, 15, 20, 25, 30, 35, 45, 50, 75, 100, 150,or 200, or 20 or 30 or 50-400 nm in diameter. Smaller particles tend tobe cleared more rapidly form the system. Therapeutic agents, includingvaccines, can be entrapped within or coupled, e.g., covalent coupled, orotherwise adhered, to nanoparticles.

Lipid- or oil-based nanoparticles, such as liposomes and solid lipidnanoparticles and can be used to can be used to deliver therapeuticagents, e.g., vaccines, described herein. Solid lipid nanoparticles forthe delivery of therapeutic agents are descripbed in Serpe et al. (2004)Eur. J. Pharm. Bioparm. 58:673-680 and Lu et al. (20060 Eur. J. Pharm.Sci. 28: 86-95. Polymer-based nanoparticles, e.g., PLGA-basednanoparticles can be used to deliver agents described herein. These tendto rely on biodegradable backbone with the therapeutic agentintercalated (with or without covalent linkage to the polymer) in amatrix of polymer. PLGA is a widely used in polymeric nanoparticles, seeHu et al. (2009) J. Control. Release 134:55-61; Cheng et al. (2007)Biomaterials 28:869-876, and Chan et al. (2009) Biomaterials30:1627-1634. PEGylated PLGA-based nanoparticles can also be used todeliver theraputic agents, see, e.g., Danhhier et al., (2009) J.Control. Release 133:11-17, Gryparis et al (2007) Eur. J. Pharm.Biopharm. 67:1-8. Metal-based, e.g., gold-based nanoparticles can alsobe used to deliver therapeutic agents. Protein-based, e.g.,albumin-based nanoparticles can be used to deliver agents describedherein. In some embodiments, a therapeutic agent can be bound tonanoparticles of human albumin.

A broad range of nanoparticles are known in the art. Exemplaryapproaches include those described in WO2010/005726, WO2010/005723WO2010/005721, WO2010/121949, WO2010/0075072, WO2010/068866,WO2010/005740, WO2006/014626, 7,820,788, 7,780,984, the contents ofwhich are incorporated herein in reference by their entirety.

Dosages

Any dosage amount (e.g., a standard dose and/or a fractional dose) of avaccine, antigen, and/or immunogen that is capable of eliciting animmune response (e.g., immunogenicity and/or broad-spectrum immunity) ina subject, e.g., when administered by a microneedle of the invention,may be used according to the methods described herein. In someembodiments, dose, e.g., the standard dose (e.g., human dose) for avaccine, an antigen, and/or an immunogen (e.g., an influenza vaccine) isbetween about 0.1 μg and about 65 μg (e.g., between about 0.1 μg andabout 10 μg, between about 0.1 μg and about 1 μg, between about 0.5 μgand about 5 μg, between about 5 μg and about 10 μg, between about 10 μgand about 20 μg, between about 20 μg and about 30 μg, between about 30μg and about 40 μg, about 40 μg and about 50 μg, about 50 μg and about65 μg, e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, or 65 μg). In some embodiments, the dose, e.g.,standard human dose, for a vaccine described herein (e.g., an influenzavaccine) is approximately between about 1 μg and about 30 μg per strain,e.g., between about 5 μg and about 30 μg per strain (e.g., about 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 μg per strain). In some embodiments, the dose,e.g., fractional dose, for a vaccine described herein (e.g., aninfluenza vaccine) is no more than 1/X, wherein X is any number, e.g.,wherein X is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more, ofthe total dose (e.g., a standard dose). It is known in the art, thatthere is clinical precedent for dose-sparing when delivering influenzavaccine to the intradermal space (e.g., Fluzone ID), and this this doseis about 9 μg per strain. Accordingly, in some embodiments the totaldosage amount of an influenza vaccine (e.g., Fluzone ID) that can bedelivered by a microneedle of the invention can be between about 5 μgand 13 μg (e.g., about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9μg, about 10 μg, about 11 μg, about 12 μg, or about 13 m).

Without wishing to be bound by theory, the total dosage amount (e.g., astandard dose) of a vaccine, antigen, and/or immunogen to beadministered by a microneedle described herein can be divided between aplurality of microneedles (e.g., within a patch), such that amicroneedle tip can comprises less than about 1% of the total dosageamount (e.g., in an array comprising about 121 microneedles), or atleast about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% or more of thetotal dosage amount. In some embodiments, an implantable microneedletip, as described herein, can comprise about 0.1 μg to about 65 μg(e.g., about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1 μg,about 1 μg to about 10 μg, about 10 μg to about 20 μg, about 20 μg toabout 30 μg, about 30 μg to about 40 μg, about 40 μg to about 50 μg,about 50 μg to about 65 μg) of a vaccine, antigen, and/or immunogen, asdescribed herein.

In some embodiments, the vaccine dosage amount loaded into a microneedlepatch can be manipulated via the concentration of antigen in theformulated solution that forms the needle tips, the volume of solutiondispensed into each needle tip, and the total number of needles (theformer two are more convenient means of varying dose). The dosagereleased into the skin is related to deployment efficiency (the portionof needle tips that are left behind in the skin after the patch isremoved), and also the release profile over time and the residence timeof the tips within the skin. Because of the continuous sloughing of skinfrom the epidermis, deeper deployment within the skin is related tolonger residence time. Therefore, it is desirable to maximize thepenetration depth of the needle tip (up to a limit defined by the depthof pain receptors within the skin, e.g., at a depth of between about 100μm and about 600 μm), and also to have the antigen spatiallyconcentrated toward the tip of the needle.

The formulations, compositions, articles, devices, and/or preparationsdescribed herein, including the implantable sustained-release tipformulation, are designed to not only sustain release of vaccine antigenover the duration, e.g., of tip retention in the dermis, but to alsomaintain stability of antigen during this period of time (e.g., at leastabout 1-2 weeks). In some embodiments, approximately 95-100% of thetotal dosage amount incorporated, e.g., in a formulation, composition,article, device, preparation, and/or microneedle described herein, canbe expected to be available for delivery, e.g., into a subject, e.g.,into a tissue of a subject, such as the skin, a mucous membrane, anorgan tissue, a buccal cavity, a tissue, or a cell membrane. Withoutbeing bound by theory, successful deployment of a microneedle into theskin is at least about 50% and can be as high as 100% of an array (e.g.,upon application at least about 50%, 60%, 70%, 80%, 90% or more (e.g.,100%) of the total number of microneedle comprising an array aresuccessfully deployed within, e.g., the skin, for controlled- orsustained-release of a vaccine antigen). In some embodiments, a portionof antigen may not be released from the silk tips during the duration ofdeployment.

Uses

The invention also provides methods for delivering a vaccine, anantigen, and/or an immunogen (e.g., an influenza vaccine) across abiological barrier (e.g., the skin). Such methods can include providinga formulation, composition, article, device, preparation, and/ormicroneedle described herein. For example, such methods can includeproviding at least one microneedle or at least one microneedle devicedescribed herein, wherein the microneedle or the microneedle devicecomprises a silk fibroin-based implantable tip having at least onevaccine, antigen, and/or an immunogen (e.g., an influenza vaccine);causing the microneedle or microneedle device to penetrate into thebiological barrier (e.g., the skin); and allowing the vaccine, antigen,and/or an immunogen to be released from the implantable tips over aperiod of at least about 4 days (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, or 14 or more days, e.g., between about 4 days and about14 days, e.g., between about 1-2 weeks, about 1-3 weeks, or about 1-4weeks). In some embodiments, the vaccine, antigen, and/or an immunogenis released into the biological barrier through the degradation and/ordissolution of the implantable microneedle tips. In some embodiments,the microneedle or microneedle device is configured to administer thevaccine, antigen, and/or an immunogen in an amount and/or a durationthat results in broad-spectrum immunity in the subject, e.g., animmunity against one or more viral antigens not present in theimplantable sustained-release tip, e.g., an immunity against a driftedstrain not present in the implantable sustained-release tip.

The invention also provides a method for providing broad-spectrumimmunity to a virus, e.g., an influenza virus, in a subject, said methodcomprising administering a vaccine (e.g., a influenza vaccine) in anamount (e.g., a dosage) and/or over a time period sufficient to resultin broad-spectrum immunity to a virus, e.g., results in an immuneresponse (e.g., a cellular immune response and/or a humoral immuneresponse) to a drifted strain of the virus, in the subject. In someembodiments, the vaccine is administered in a composition for thecontrolled- or sustained-release of the vaccine (e.g., for thecontrolled- or sustained-release of one or more viral antigens asdescribed herein). In some embodiments, the vaccine is administered by adevice for the controlled- or sustained-release of the vaccine (e.g.,for the controlled- or sustained-release of one or more viral antigensas described herein). The vaccine can be administered into a subject,e.g., in to a tissue or cavity of the subject chosen from skin, mucosa,organ tissue, muscle tissue or buccal cavity.

In some embodiments, the methods described herein comprise administeringa in an amount (e.g., a dosage) and/or over a time period sufficient toresult in one or more of: (i) exposure in the subject to one or moreantigens in the vaccine in an amount and/or period of time to result inbroad spectrum immunity, e.g., to result in an immune response (e.g., acellular immune response and/or a humoral immune response) to a driftedstrain of the virus, in the subject; or (ii) a level of one or moreantigens in the subject that is substantially steady, e.g., about 20%,15%, 10%, 5%, or 1% to an amount, e.g., minimum amount, needed to resultin an immune response (e.g., a cellular immune response and/or a humoralimmune response) to the one or more antigens. In some embodiments, thecomposition or device for the controlled- or sustained-release of thevaccine is chosen from: a microneedle (e.g., a microneedle device, e.g.,a microneedle patch, e.g., as described herein), an implantable device(e.g., a pump, e.g., a subcutaneous pump), an injectable formulation, adepot, a gel (e.g., a hydrogel), an implant, or a particle (e.g., amicroparticle and/or a nanoparticle).

In some embodiments, the vaccine is administered, e.g., released by thecomposition or device for the controlled- or sustained-release of thevaccine, e.g., into the subject, in order to maintain a vaccine dosage(e.g., an antigen concentration) for a period of time sufficient toresult in broad spectrum immunity, e.g., to result in an immune response(e.g., a cellular immune response and/or a humoral immune response) to adrifted strain of the virus, in the subject (e.g., wherein the period oftime is about 1 to 21 days, e.g., about 5 to 10 days or about 5 to 7days, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, or 21 days). The composition or device for thecontrolled- or sustained-release of the vaccine can maintain antigenrelease and/or level in the subject over a sustained period of time. Insome embodiments the composition or device for the controlled- orsustained-release of the vaccine maintains a continuous ornon-continuous antigen release into the subject over a sustained periodof time. The vaccine can administered, e.g., released by the compositionor device for the controlled- or sustained-release, over a period oftime comprising at least about one week, e.g., about 1-2 weeks, about1-3 weeks, or about 1-4 weeks. In some embodiments, the vaccine isadministered, e.g., released by the composition or device for thecontrolled- or sustained-release, over a period of time comprising atleast about 4 days (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 days, or more, e.g., between about 4 days andabout 2 weeks, between about 4 days and about 1 week).

The vaccine can be administered in a dosage comprising between about 0.1μg and about 65 μg per strain, e.g., 0.2 μg and about 50 μg per strain(e.g., about each of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, or 65 μg per strain). In some embodiments, atleast about 1% of the dosage of the vaccine (e.g., at least about 0.5%to about 10%, at least about 5% to about 15% at least about 10% to about20% of the dosage), e.g., released by the composition or device for thecontrolled- or sustained-release of the vaccine, e.g., into the subject,is maintained over a period of time comprising at least about 4 days(e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or more,e.g., between about 4 days and about 2 weeks, between about 4 days andabout 1 week).

In some embodiments, the vaccine is administered, e.g., released by thecomposition or device for the controlled- or sustained-release, in aplurality of fractional doses of a total dose (e.g., a standard dose)over a time period, e.g., such that an immune response and/orbroad-spectrum immunity is achieved, wherein the amount of the vaccineadministered in each of the fractional doses is no more than 1/X,wherein X is any number, e.g., wherein X is 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50,60, 70, 80, 90, or 100 or more, of the total dose (e.g., a standarddose) of the vaccine.

In some embodiments, the vaccine is administered, e.g., released by thecomposition or device for the controlled- or sustained-release of thevaccine, e.g., into the skin of the subject, in a plurality of dosesequivalent to a percentage of a total dose (e.g., a percentage of astandard dose) over a time period, e.g., such that broad-spectrumimmunity is achieved, wherein the amount of the vaccine administered ineach of the plurality of doses is about X %, wherein X is any number,e.g., wherein X is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150,175, 200, 300, 400, or 500 or more, of the total dose (e.g., a standarddose) of the vaccine.

The vaccine can be administered according to any of the methodsdescribed herein such that broad-spectrum immunity is achieved, e.g.,such that an immune response, e.g., a cellular immune and/or humoralimmune response to a drifted strain is achieved.

Without wishing to be bound by theory, a subject exposed to and/orinfected with a first influenza virus can develop an immune response(e.g., a cellular immune and/or humoral immune response) resulting inthe creation of an antibody against that first influenza virus. Asantigenic changes (e.g., mutations) accumulate in the first influenzavirus over time, the subject's antibodies created against the firstinfluenza virus may no longer recognize the drifted virus (e.g., theantigenically different strain). Using the methods, dosage regimens,microneedles, and microneedle devices described herein, broad-spectrumimmunity can be conferred to a subject exposed to, infected with, and/orat risk of infection with an influenza virus. Further, using themethods, dosage regimens, microneedles, and microneedle devicesdescribed herein, improved immunogenicity and/or broad-spectrum immunitycan be conferred to a subject, e.g., as compared to traditional burstrelease administration of vaccine. For example, improved immunogenicityand/or broad-spectrum immunity detectable in a subject can be greater(e.g., 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold or moregreater) as compared to traditional burst release administration ofvaccine, e.g., the administration of a single-dose or a bolusadministration of the vaccine.

In some embodiments, the implantable sustained-release tip or thevaccine comprises a first influenza strain and administration of a doseof the first influenza strain (e.g., a first influenza A, B, C, and/or Dstrain as described herein) to the subject results in the development ofbroad-spectrum immunity to a second influenza strain (e.g., a driftedinfluenza A, B, C, and/or D strain as described herein) not present inthe implantable sustained-release tip or the vaccine.

In some embodiments, the subject (e.g., the human subject) is apediatric subject, an adult subject, or an elderly subject. The subjectmay have been exposed to, infected with, and/or at risk of infectionwith an influenza virus (e.g., a particular strain of an influenzavirus). Such a risk may be due to the health status or age of thesubject and/or travel to a region where a particular strain of influenzavirus is prevalent.

In some embodiments, the invention provides methods of providing acontrolled- or sustained-release of a vaccine in a subject. Thecontrolled- or sustained-release of the vaccine can achieve an improvedimmunogenicity and/or broad-spectrum immunity, as compared totraditional burst release administration of vaccine. Without wishing tobe bound by theory, an method of administering a vaccine describedherein and/or a controlled- or sustained-release rate, e.g., by acomposition and/or a microneedle described herein, that mimics thenatural exposure pattern of a subject (e.g., a human subject) to a viruscan provide enhanced immunity and/or broad-spectrum immunity to asubject, as compared to traditional single-dose vaccine administrationmodalities.

In some embodiments, a desired amount of at least one vaccine, antigen,and/or immunogen (e.g., an influenza vaccine) can be released from themicroneedle (e.g., implantable mironeedle tip) described herein in asustained manner over a pre-defined period of time. In some embodiments,at least about 5% of a vaccine, an antigen, and/or an immunogen (e.g.,an influenza vaccine), e.g., at least about 10%, at least about 15%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, or at least about 97%, about 98%,or about 99%, or 100% of the vaccine, antigen, and/or an immunogen(e.g., an influenza vaccine), can be released from the microneedle(e.g., implantable microneedle tips) over a pre-defined period of time.In such embodiments, the desired amount (e.g., a dose, such as astandard dose of a vaccine) of the vaccine, antigen, and/or immunogen(e.g., an influenza vaccine) can be released from the microneedle overseconds, minutes, hours, months and/or years. In some embodiments, thedesired amount (e.g., a dose, such as a standard dose of a vaccine) ofthe vaccine, antigen, and/or immunogen (e.g., an influenza vaccine) canbe released from the microneedle upon insertion into a biologicalbarrier, e.g., within 5 seconds, within 10 seconds, within 30 seconds,within 1 minute, within 2 minutes, within 3 minutes, within 4 minutes,within 5 minutes or longer. In some embodiments, the desired amount(e.g., a dose, such as a standard dose of a vaccine) of the vaccine,antigen, and/or immunogen (e.g., an influenza vaccine) can be releasedfrom the microneedle over a period of at least about 1 hour, at leastabout 2 hours, at least about 3 hours, at least about 6 hours, at leastabout 12 hours, at least about 1 day, at least about 2 days, at leastabout 3 days, at least about 4 days, at least about 5 days, at leastabout 6 days, at least about 1 week, at least about 2 weeks, at leastabout 1 month, at least about 2 months, at least about 3 months, atleast about 6 months or longer. In some embodiments, the desired amount(e.g., a dose, such as a standard dose of a vaccine) of the vaccine,antigen, and/or immunogen (e.g., an influenza vaccine) can be releasedfrom the microneedle over about 1 year or longer.

In some embodiments, the invention provides methods for enhancing animmune response to a virus in a subject. In some embodiments, thepresence of a cell-mediated immunological response can be determined byany art-recognized methods, e.g., proliferation assays (CD4+ T cells),CTL (cytotoxic T lymphocyte) assays (see Burke, supra; Tigges, supra),or immunohistochemistry with tissue section of a subject to determinethe presence of activated cells such as monocytes and macrophages afterthe administration of an immunogen. One of skill in the art can readilydetermine the presence of humoral-mediated immunological response in asubject by any well-established methods. For example, the level ofantibodies produced in a biological sample such as blood can be measuredby western blot, ELISA or other methods known for antibody detection. Insome embodiments, an elevated hemagglutination inhibition (HAI) antibodytiter is detectable in the blood of the subject for the duration of acomplete flu season post immunization.

In some embodiments, the immune response and/or the broad-spectrumimmunity is a cellular immune and/or humoral immune response comprising:(i) an elevated hemagglutination inhibition (HAI) antibody titerdetectable in the blood of the subject, e.g., detectable at least 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, and/or 30-weeks or more post immunization; (ii)an elevated anti-influenza IgG titer detectable in the blood of thesubject, e.g., detectable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11and/or 12-months or more post immunization; and/or (iii) a level ofantibody secreting plasma cells (ASC) against the virus, e.g., theinfluenza virus, detectable in the bone marrow of the subject, e.g.,detectable at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and/or34-weeks or more post immunization. In some embodiments, the elevatedHAI antibody titer is to a drifted influenza A, B, C, and/or D strain.In some embodiments, the elevated anti-influenza IgG titer is to adrifted influenza A, B, C, and/or D strain. In some embodiments, theimmune response is a cellular immune response comprising an increase inthe level of IFNγ secreting cell in the blood of the subject, e.g., atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12-weeks or more postimmunization, e.g., by a microneedle described herein.

In some embodiments, the elevated HAI antibody titer, the elevatedanti-influenza IgG titer, the level of antibody secreting plasma cells(ASC) against the virus, and/or the level of IFNγ secreting cellsdetectable in the subject is greater (e.g., 1-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold,12-fold, 13-fold, 14-fold, or 15-fold or more greater) as compared tothe administration of a single-dose or a bolus administration of thevaccine.

In some embodiments, broad-spectrum immunity can be characterized bymeasuring the percent seroconversion in a subject. For example,broad-spectrum immunity can comprise a percent seroconversion, e.g.,based on the elevated HAI antibody titer detectable in the blood of thesubject, e.g., at 6-month post immunization greater than about 20%(e.g., 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95% or more, e.g., 100%). Such a level of seroconversionassociated with broad-spectrum immunity conferred by using the methods,dosage regimens, microneedles, and microneedle devices described hereincan be greater (e.g., 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or15-fold or more greater) as compared to a level of seroconversionobtained by traditional burst release administration of vaccine, e.g.,the administration of a single-dose or a bolus administration of thevaccine.

Combination Therapies

The microneedles and microneedle devices (e.g., microneedle patches)described herein may be manufactured by precision filling of eachindividual microneedle tip to enable different patterns of vaccinedelivery, dosing schemes, and combination administration of a vaccinewith an additional therapeutic agent. The methods of immunization,vaccine delivery, and dosing described herein may comprise combinationadministration of a vaccine with an additional therapeutic agent. Insome embodiments, an additional therapeutic agent may be formulated inthe same tip as a vaccine. In some embodiments, an additionaltherapeutic agent may be formulated with the vaccine. For example,adjuvants to boost immune response to co-delivered antigen could bedelivered in the same microneedle tip and/or vaccine. Without wishing tobe bound by theory, such a combination therapy could include adjuvantsto drive stronger cellular immune responses and/or mucosal responses.Moreover, additional influenza antigens could be delivered forheterologous “prime/boost-like” immunization, e.g., primary immunizationwith an HA antigen from various influenza strains and a boost (e.g.,provided via controlled- or sustained-release or distinct kineticpattern from “prime”) with a different antigen (e.g., a drifted strain,a hemagglutinin stem, m2e protein, or NA).

Formulation compatibility may limit whether two given therapeutic agentscan be co-formulated to be dispensed into the same needle tip. In caseco-formulation is not possible, the manufacturing process can be adaptedin order to dispense a first formulation into a portion of the needlearray and then dispense a second formulation into a different portion ofthe needle array. Different formulations can also receive differentprocess treatments after filling. For instance, if the first formulationwill be for controlled- or sustained-release and the silk will berendered less soluble via water annealing, while the second formulationwill be for burst release with no annealing, the second formulation canbe dispensed after the annealing step. The manufacturing approach isflexible so other process sequences are possible.

In some embodiments, the invention also provides methods for combinationtherapies, wherein a microneedle or microneedle device of the inventioncan be fabricated to administer at least one additional therapeuticagent. Various forms of a therapeutic agent can be used which arecapable of being released from the microneedles described herein intoadjacent tissues or fluids upon administration to a subject. In someembodiments, an additional therapeutic agent can be included within thebase layer and/or within the implantable tip.

Examples of additional therapeutic agents that can be used according tothe methods of the invention, e.g., incorporated into a microneedle ofthe invention, e.g., during fabrication, include steroids and esters ofsteroids (e.g., estrogen, progesterone, testosterone, androsterone,cholesterol, norethindrone, digoxigenin, cholic acid, deoxycholic acid,and chenodeoxycholic acid), boron-containing compounds (e.g.,carborane), chemotherapeutic nucleotides, drugs (e.g., antibiotics,antivirals, antifungals), enediynes (e.g., calicheamicins, esperamicins,dynemicin, neocarzinostatin chromophore, and kedarcidin chromophore),heavy metal complexes (e.g., cisplatin), hormone antagonists (e.g.,tamoxifen), non-specific (non-antibody) proteins (e.g., sugaroligomers), oligonucleotides (e.g., mRNA sequences or antisenseoligonucleotides that bind to a target nucleic acid sequence), peptides,proteins, antibodies, photodynamic agents (e.g., rhodamine 123),radionuclides (e.g., 1-131, Re-186, Re-188, Y-90, Bi-212, At-211, Sr-89,Ho-166, Sm-153, Cu-67 and Cu-64), toxins (e.g., ricin), andtranscription-based pharmaceuticals.

Exemplary Kits

In certain embodiments, the invention relates to a package or kitcomprising a microneedle described herein (e.g., a microneedle includinga vaccine, antigen, and/or an immunogen as described herein, such as aninfluenza virus). In some embodiments, the invention relates to apackage or kit comprising a vaccine described herein (e.g., a vaccine,antigen, and/or an immunogen as described herein, such as an influenzavirus). In some embodiments, the kit can further comprise an additionaltherapeutic for combination therapy with the microneedle. In someembodiments, the kits can further comprise a disinfectant (e.g., analcohol swab). In some embodiments, such packages, and kits describedherein can be used for vaccination purposes, e.g., to achievebroad-spectrum immunity in a subject as described herein.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Example 1. Sustained Intradermal Delivery of Influenza Vaccine GeneratesImproved Cellular Responses and Stronger, Longer-Lasting AntibodyResponses

Balb/c mice were immunized with FluzoneHD at 0.5 ug/strain either byintramuscular injection or by intradermal injections of fractional dosesfor a total of 10 days. Anti-flu IgG responses were measured by ELISA.As shown in FIGS. 1A-1B, 10 day controlled- or sustained-release ofvaccine results in significantly higher titers compared to equivalentintramuscular injections. Haemagglutination inhibition titers above 40are known correlates of protection against infection. HAI titers forA/HongKong/H3N2 and B/Brisbane were measured at day 28 and 56 postimmunization. At both time points, higher HAI titers were observed uponintradermal controlled- or sustained-release than IM injections (FIGS.1C-1D) indicating higher correlates of protection by controlled- orsustained-release. T cell responses following vaccination were measuredat week 12 by ELISPOT. A trend towards increased IFNγ+ cells inperipheral blood was observed upon sustained intradermal vaccinedelivery when compared to IM injections (FIGS. 1E-1F). Taken together,these results indicate that sustained delivery of a vaccine againstinfluenza results in stronger humoral and cellular responses thanequivalent dose delivered by conventional intramuscular injections.

Example 2. Controlled- or Sustained-Release Microneedle Formulation andFabrication

Trivalent influenza vaccine (TIV) (Fluzone® High-Dose, 2017-18 formula,Sanofi-Pasteur, Swiftwater, Pa.) was prepared for microneedle devicefabrication through processing to remove excess detergent and toconcentrate HA antigen. 10 doses of TIV were run serially throughdetergent removal columns (Pierce™ Detergent Removal Spin Column, 2 mL,ThermoFisher 87778) to remove Triton X-100 (octyl phenol ethoxylate)detergent, a byproduct of manufacturing used to split influenza virus.An aliquot of material was collected and reserved for analysis via sizeexclusion chromatography (HPLC-SEC) to confirm absence of free detergentpeaks. The remaining material was concentrated in 10 kDa spin filters(Amicon Ultra 0.5 mL, Fischer Sci 501096) through up to 3 10-minutespins at 15000 rpm. An aliquot of material was run on HPLC-SEC todetermine concentration of flu antigens against initial vaccine.Comparison of area-under-the-curve (AUC) for pre-concentration andpost-concentration material was used to determine the concentration ofthe processed antigen stock. 100 uL of stock was mixed with 85.6 uL ofsilk fibroin (60 MB) and 64.4 uL of Milli-Q water to generate a 5% (w/v)silk fibroin, 192 ug/mL HA (per strain) solution to be printed intomicroneedle molds.

Tip Filling: 20 nL of formulation was printed using vision-guideddispensing (Biodot AD3420) into a PDMS microneedle mold.

Tip Fill Inspection: Printing was visually assessed understereomicroscope for defects, including misaligned prints, incompletelyfilled needle cavities, and foreign debris.

Tip Dry: Filled microneedle molds were dried under controlled 20% RHconditions overnight (14-20 hours).

Tip Anneal: Dried tips were water annealed at 37C for four hours,through placement of molds in a vacuum desiccator filled with Milli-Qwater, applying vacuum for 5 minutes, then closing vacuum valve andmoving desiccator to 37C incubator.

Tip Dry: after annealing tips were again dried under controlled 20% RHconditions overnight (14-20 hours).

Base Filling: A solution of 40% (w/v) hydrolyzed gelatin (Gelita) and10% (w/v) sucrose (Sigma-Aldrich) was pipetted onto microneedle moldsand filled via centrifugation at 3900 rpm for 2 minutes.

Base Fill Inspection: Base filling is also assessed visually bystereomicroscope for the appearance of needle cavities that were notentirely filled. Re-filling and re-centrifugation is performed if lackof fill is observed.

Base Drying: Base solution is dried under controlled 20% RH conditionsovernight (14-20 hours).

Backing Apply: Whatman 903 cards were punched into 12 mm discs andapplied to pre-wetted (10 uL Milli-Q water) dried gelatin base.

Backing Dry: Devices were dried under controlled 20% RH conditions for 2hours before demolding.

Demolding: Devices were manually removed from microneedle molds bycarefully bending the mold away from the device while holding devicestationary.

Demold Inspect: Devices were inspected for complete demolding understereomicroscope; incompletely demolded devices were discarded.

Example 3. Immunization Via Controlled- or Sustained-Release SilkMicroneedles Improves Humoral and Cellular Responses

Balb/c mice were immunized by either intramuscular injections (IM) ormicroneedles that can sustain release the vaccine in the skin (MN).Following immunization, the anti-flu IgG titers were measured by ELISA.As shown in FIGS. 2A-2B, a 3-5 fold increase in titers is observed for 4months and 6 months post immunization with MN compared to IM injection.HAI titers for the 3 strains, A/Hong Kong/H3N2, A/Michigan/H1N1 andB/Brisbane were measured at months 1, 2, 3, 4, and 6 post immunization(FIGS. 2C-2H). Significantly higher HAI titers were observed with MNwith complete seroconversion maintained at month 4 compared to IMinjection (FIGS. 2C and 2E). Significantly higher HAI titers wereobserved with MN with complete seroconversion maintained at month 6compared to IM injection for the two A strains and a trend towardsimproved seroconversion for the B lineage (FIGS. 2D, 2F, and 2H). IFNγcellular responses in peripheral blood was also significantly higherupon MN delivery of vaccine than IM delivery (FIG. 2E-2F). At week 4post vaccination, significantly higher vaccine specific IFNγ+ cells inperipheral blood was determined by ELISPOT for MN delivery (FIG. 2I-2J).These results demonstrate the enhanced immunogenicity of vaccinationpossible though microneedle delivery.

Example 4. Controlled- or Sustained-Release of Influenza Vaccine Resultsin an Immune Response, e.g., Humoral and Cellular Immune Responses,Against Drifted Strains of Influenza Controlled- or Sustained-Release ofInfluenza Vaccine Generates Higher HAI Titers Against Drifted H3N2Strain of Influenza

Balb/c mice were immunized with FluzoneHD at 0.5 ug/strain either byintramuscular injection or by intradermal injections of fractional dosesfor a total of 10 days or by application of the MIMIX (microneedle, MN)patch. HAI titers for A/Switzerland/H3N2/2013 (a strain that was notincluded in the vaccine) were measured at month 4 and 5 (days 120 and150) post immunization respectively. As shown in FIGS. 6A-6B, 10 daycontrolled- or sustained-release of vaccine (SR) results insignificantly higher titers to the drifted strain compared to equivalentintramuscular injections. Haemagglutination inhibition titers above 40are known correlates of protection against infection. MIMIX (MN)delivery also showed a trend towards increased HAI titers with 3 out of5 mice achieving a HAI titer of 40 compared to no animals in the IMimmunized group at month 4 post immunization indicating highercorrelates of protection by controlled- or sustained-release.

Controlled- or Sustained-Release of Influenza Vaccine Generates HigherHAI Titers Against Drifted H1N1 Strain of Influenza

Balb/c mice were immunized with FluzoneHD at 0.5 ug/strain either byintramuscular injection or by intradermal injections of fractional dosesfor a total of 10 days or by application of the MIMIX patch. HAI titersfor A/California/7/2009/H1N1 (a strain that was not included in thevaccine) were measured at month 6 (day 180) post immunization. As shownin FIG. 8, 10 day controlled- or sustained-release of vaccine results insignificantly higher titers compared to equivalent intramuscularinjections to the drifted vaccine strain. Haemagglutination inhibitiontiters above 40 are known correlates of protection against infection.MIMIX (MN) delivery also showed a trend towards increased HAI titerswith 3 out of 5 mice achieving a HAI titer of 40 compared to no responsein animals in the IM immunized group at month 4 post immunizationindicating higher correlates of protection by controlled- orsustained-release.

Controlled- or Sustained-Release of Influenza Vaccine Generates HigherHAI Titers Against B Lineage Strain not Included in the Vaccine

Balb/c mice were immunized with FluzoneHD at 0.5 ug/strain either byintramuscular injection or by intradermal injections of fractional dosesfor a total of 10 days or by application of the MIMIX patch. HAI titersfor B/Phuket were measured at week 7 (day 49) post immunization.B/Phuket belongs to the Yamagata lineage that was not included in thevaccine. As shown in FIG. 10, sustained vaccine release from MIMIXshowed a trend towards increase in HAI titers to this B lineage.

Controlled- or Sustained-Release of Influenza Vaccine Generates MoreLong-Lived Plasma Cells in the Bone Marrow Against Both Vaccine Includedand Drifted H3N2 Strains of Influenza

Balb/c mice were immunized with FluzoneHD at 0.5 ug/strain either byintramuscular injection or by intradermal injections of fractional dosesfor a total of 10 days. At month 8 (day 240) post immunization, animalswere sacrificed and the cells from the bone marrow were isolated. A Bcell ELISPOT was performed (following manufacturer's instructions,Immunospot) to measure antibody secreting plasma cells (ASC) against thevaccine included strain (A/Hong Kong/H3N2) and drifted strain(A/Switzerland/H3N2). As shown in FIGS. 7A-7B, fractional dosing of thevaccine over 10 days (SR) resulted in significantly higher number ofboth vaccine-specific and drifted strain specific ASCs.

Sustained Release of Influenza Vaccine Generates More Long-Lived PlasmaCells in the Bone Marrow Against Both Vaccines Included and Drifted H1N1Strains of Influenza

Balb/c mice were immunized with FluzoneHD at 0.5 ug/strain either byintramuscular injection or by intradermal injections of fractional dosesfor a total of 10 days. At month 8 (day 240) post immunization, animalswere sacrificed and the cells from the bone marrow were isolated. A Bcell ELISPOT was performed (following manufacturer's instructions,Immunospot) to measure antibody secreting plasma cells (ASC) against thevaccine included strain (A/Michigan/H1N1) and drifted strain(A/California/H1N1). As shown in FIGS. 9A-9B, fractional dosing of thevaccine over 10 days (SR) showed a trend towards increase in bothvaccine-specific and drifted strain specific ASCs.

Significantly Higher Number of Both ASC Against the Vaccine IncludedB/Lineage Strain (B/Brisbane) and to the B/Yamagata Lineage (B/Phuket)

Balb/c mice were immunized with FluzoneHD at 0.5 ug/strain either byintramuscular injection or by intradermal injections of fractional dosesfor a total of 10 days. At month 8 (day 240) post immunization, animalswere sacrificed and the cells from the bone marrow were isolated. A Bcell ELISPOT was performed (following manufacturer's instructions,Immunospot) to measure antibody secreting plasma cells (ASC) against thevaccine included B/lineage strain (B/Brisbane) and to the B/Yamagatalineage (B/Phuket). As shown in FIGS. 11A-11B, fractional dosing of thevaccine over 10 days (SR) resulted in significantly higher number ofboth vaccine-specific and drifted strain specific ASCs.

CONCLUSION

Controlled- or sustained-release leads to increased plasma cells andprotective HAI titers against both vaccine and drifted influenzaviruses, suggesting stronger and broader protection. Table 1, below,indicates the percent (%) seroconversion corresponding to the data inFIGS. 9, 11, and 13. Taken together, these results indicate thatsustained delivery of a vaccine against influenza results in strongerHAI titers to drifted (non-vaccine) strains than equivalent dosedelivered by conventional intramuscular injections. Table 2, below,indicates the fold increase post sustained vaccine release (SR) overintramuscular injection (IM) in the count of long-lived plasma cells inthe bone marrow specific for vaccine included and drifted strainsquantified in FIGS. 10, 12, and 14. Taken together, these resultsindicate that controlled- or sustained-release of the vaccine againstinfluenza results in durable presence of antibody secreting plasma cellsto drifted (non-vaccine) strains than equivalent dose delivered byconventional intramuscular injections.

TABLE 1 Percent (%) seroconversion based on HAI titers to vaccineincluded and drifted strains Sustained Vaccine Micronee- %Seroconversion Intramuscular Release dle per Strain Injection (IM) (SR)(MN) H1N1 A/Michigan (Vac) 20 80 80 A/California (Drift) 20 80 60 H3N2A/Hong Kong (Vac) 20 80 100 A/Switzerland* (Drift) 0 80 60 B B/Brisbane(Vac) 0 — 40 B/Phuket{circumflex over ( )} (Drift) 20 — 66 *at month 4,{circumflex over ( )}at week 7

TABLE 2 Fold increase post sustained vaccine release (SR) overintramuscular injection (IM) Strain Fold increase, SR vs IM H1N1A/Michigan (Vac) 5.4 A/California (Drift) 7.9 H3N2 A/Hong Kong (Vac)10.9 A/Switzerland (Drift) 5.5 B B/Brisbane (Vac) 7.9 B/Phuket (Drift)13.5

EQUIVALENTS

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the appended claims.

What is claimed is:
 1. A microneedle comprising: (i) a backing, (ii) adissolvable base comprising one, two, three, four or more of gelatin,polyethylene glycol (PEG), sucrose, carboxymethylcellulose (CMC),polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronate,maltose, and methyl cellulose applied to the backing, (iii) animplantable sustained-release tip comprising a silk fibroin applied tothe dissolvable base, wherein the microneedle is configured to implantthe sustained-release tip into the skin of a subject, e.g., a humansubject, at a depth (e.g., a max penetration depth of the distal part oftip) of between about 100 μm and about 600 μm, wherein thesustained-release tip comprises a silk fibroin, e.g., a regenerated silkfibroin and/or a recombinant silk fibroin, wherein the sustained-releasetip further comprises a vaccine (e.g., an influenza vaccine) in anamount sufficient to induce an immune response, e.g., a humoral immuneresponse and/or a cellular immune response.
 2. The microneedle of claim1, wherein the dissolvable base comprising one of gelatin, polyethyleneglycol (PEG), sucrose, carboxymethylcellulose (CMC),polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronate,maltose, and methyl cellulose.
 3. The microneedle of claim 1, whereinthe dissolvable base comprising two of gelatin, PEG, sucrose, CMC, PVP,PVA, hyaluronate, maltose, and methyl cellulose.
 4. The microneedle ofclaim 1, wherein the dissolvable base comprising three of gelatin, PEG,sucrose, CMC, PVP, PVA, hyaluronate, maltose, and methyl cellulose. 5.The microneedle of claim 1, wherein the dissolvable base comprising fourof gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate, maltose, andmethyl cellulose.
 6. The microneedle of claim 1, wherein the dissolvablebase comprising five of gelatin, PEG, sucrose, CMC, PVP, PVA,hyaluronate, maltose, and methyl cellulose.
 7. The microneedle of claim1, wherein the dissolvable base comprising six of gelatin, PEG, sucrose,CMC, PVP, PVA, hyaluronate, maltose, and methyl cellulose.
 8. Themicroneedle of claim 1, wherein the dissolvable base comprising seven ofgelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate, maltose, and methylcellulose.
 9. The microneedle of claim 1, wherein the dissolvable basecomprising gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate, maltose,and methyl cellulose.
 10. The microneedle of any one of the precedingclaims, wherein the dissolvable base comprising gelatin and sucrose. 11.The microneedle of any one of the preceding claims, wherein thedissolvable base comprises CMC.
 12. The microneedle of any one of thepreceding claims, wherein the dissolvable base comprises PVP.
 13. Themicroneedle of any one of the preceding claims, wherein the dissolvablebase comprises PVA.
 14. The microneedle of any one of the precedingclaims, wherein the dissolvable base comprises PVP and PVA.
 15. Themicroneedle of any one of the preceding claims, wherein the dissolvablebase comprises PVP, PVA, and sucrose.
 16. The microneedle of any one ofthe preceding claims, wherein the dissolvable base does not comprisepoly(acrylic acid) (PAA).
 17. The microneedle of any one of thepreceding claims, wherein the implantable sustained-release tip isconfigured to release a vaccine into the skin of the subject over aperiod of time comprising at least about 4 days (e.g., about 4, 5, 6, 7,8, 9, 10, 11, 12, 13, or 14 or more days, e.g., between about 4 days andabout 14 days, e.g., between about 1-2 weeks, about 1-3 weeks, or about1-4 weeks).
 18. The microneedle of any one of the preceding claims,wherein the implantable sustained-release tip is configured to release avaccine into the skin of the subject over a period of time comprisingabout 1 week to about 2 weeks (e.g., about 7, 8, 9, 10, 11, 12, 13, or14 days).
 19. The microneedle of any one of the preceding claims,wherein immune response is a humoral immune response comprises: (i) anelevated hemagglutination inhibition (HAI) antibody titer detectable inthe blood of the subject, e.g., detectable at least 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, and/or 16-weeks post immunization; and/or (ii)an elevated anti-influenza IgG titer detectable in the blood of thesubject, e.g., detectable at least 1, 2, 3, 4, and/or 5-months postimmunization.
 20. The microneedle of any one of the preceding claims,wherein an elevated hemagglutination inhibition (HAI) antibody titer isdetectable in the blood of the subject for the duration of a completeflu season post immunization.
 21. The microneedle of any one of thepreceding claims, wherein immune response is a cellular immune responsecomprising an increase in the level of IFNγ secreting cell in the bloodof the subject, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or12-weeks post immunization.
 22. The microneedle of any one of thepreceding claims, wherein the dissolvable base comprises between about10% and about 70% gelatin (e.g., hydrolyzed gelatin) (e.g., about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, or about 70%gelatin).
 23. The microneedle of any one of the preceding claims,wherein the dissolvable base comprises between about 1% and about 35%sucrose (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about25%, about 30%, or about 35% sucrose).
 24. The microneedle of any one ofthe preceding claims, wherein the dissolvable base comprises betweenabout 1% and about 35% CMC (e.g., about 1%, about 2%, about 3%, about4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about15%, about 20%, about 25%, about 30%, or about 35% CMC).
 25. Themicroneedle of any one of the preceding claims, wherein the dissolvablebase comprises between about 10% and about 70% PVP (e.g., about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, or about 70%PVP).
 26. The microneedle of any one of the preceding claims, whereinthe dissolvable base comprises between about 1% and about 35% PVA (e.g.,e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%,about 30%, or about 35% PVA).
 27. The microneedle of any one of thepreceding claims, wherein the dissolvable base comprises between about1% and about 75% (e.g., about 1%, about 2%, about 3%, about 4%, about5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about20%, about 25%, about 30%, or about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, or about 75%hyaluronate).
 28. The microneedle of any one of the preceding claims,wherein the dissolvable base comprises between about 1% and about 75%(e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%,about 30%, or about 35%, about 40%, about 45%, about 50%, about 55%,about 60%, about 65%, about 70%, or about 75% maltose).
 29. Themicroneedle of any one of the preceding embodiments, wherein thedissolvable comprises between about 1% and about 75% (e.g., about 1%,about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%,about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, orabout 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, or about 75% methyl cellulose).
 30. The microneedle ofany one of the preceding claims, wherein the dissolvable base comprisesabout 40% hydrolyzed gelatin and about 10% sucrose w/v.
 31. Themicroneedle of any one of the preceding claims, wherein the dissolvablebase comprises up to about 50% w/v of PVP (e.g., PVP of 10 kD MW). 32.The microneedle of any one of the preceding claims, wherein thedissolvable base comprises up to about 20% PVA (e.g., 87% hydrolyzed PVAat 13 kD MW).
 33. The microneedle of any one of the preceding claims,wherein the dissolvable base comprises CMC at up to about 10%.
 34. Themicroneedle of any one of the preceding claims, wherein the dissolvablebase comprises about 1% CMC (e.g., low-viscosity CMC).
 35. Themicroneedle of any one of the preceding claims, wherein the dissolvablebase comprises about 30% PVP and about 10% PVA.
 36. The microneedle ofany one of the preceding claims, wherein the dissolvable base comprisesabout 37% PVP, about 5% PVA, and about 15% sucrose.
 37. The microneedleof any one of the preceding claims, wherein the implantablesustained-release tip comprises silk fibroin at about 1% w/v to about10% w/v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w/v, or a silkfibroin having a molecular weight distribution according to FIG. 5, or,comprises silk fibroin in an amounta between about 20 μg to about 245μg, e.g., per 121 microneedle array).
 38. The microneedle of any one ofthe preceding claims, wherein the implantable sustained-release tipcomprises a vaccine formulated in a 1% w/v to about 10% w/v (e.g., about1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w/v) of 10 MB silk fibroin solution,or a silk fibroin solution according to FIG.
 5. 39. The microneedle ofany one of the preceding claims, wherein the implantablesustained-release tip comprises a vaccine formulated in a 1% w/v toabout 10% w/v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w/v) of 60MB silk fibroin solution, or a silk fibroin solution according to FIG.5, e.g., a 100 kDa to 200 kDa (e.g., about 153 kDa) silk fibroinsolution.
 40. The microneedle of any one of the preceding claims,wherein the implantable sustained-release tip comprises a vaccineformulated in a 1% w/v to about 10% w/v (e.g., about 1, 2, 3, 4, 5, 6,7, 8, 9, or 10% w/v) of 120 MB silk fibroin solution, or a silk fibroinsolution according to FIG. 5, e.g., a 70 kDa to 150 kDa (e.g., about 100kDa) silk fibroin solution.
 41. The microneedle of any one of thepreceding claims, wherein the implantable sustained-release tipcomprises a vaccine formulated in a 1% w/v to about 10% w/v (e.g., about1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w/v) of 180 MB silk fibroin solution,or a silk fibroin solution according to FIG. 5, e.g., a 36 kDa to 100kDa (e.g., about 71 kDa) silk fibroin solution.
 42. The microneedle ofany one of the preceding claims, wherein the implantablesustained-release tip comprises a vaccine formulated in a 1% w/v toabout 10% w/v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w/v) of 480MB silk fibroin solution, or a silk fibroin solution according to FIG.5, e.g., a 1 kDa to 60 kDa (e.g., about 16 kDa) silk fibroin solution.43. The microneedle of claim 39, wherein the implantablesustained-release tip comprises a 5% wt/vol 60 MB silk fibroin solution.44. The microneedle of any one of the preceding claims, wherein theimplantable sustained-release tip comprises a standard dose of avaccine.
 45. The microneedle of any one of the preceding claims, whereinthe standard dose of the vaccine (e.g., influenza vaccine) comprisesbetween about 0.1 μg and about 65 μg per strain, e.g., 0.2 μg and about50 μg per strain (e.g., about each of 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, or 65 μg per strain).
 46. The microneedle ofclaim 44 or 45, wherein the implantable sustained-release tip comprisesat least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% or more ofthe standard dose.
 47. The microneedle of any one of claims 44-46,wherein the implantable sustained-release tip comprises about 0.1 μg toabout 65 μg of vaccine (e.g., about 0.1 μg, about 0.2 μg, about 0.3 μg,about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg,about 0.9 μg, about 1 μg, about 1 μg to about 10 μg, about 10 μg toabout 20 μg, about 20 μg to about 30 μg, about 30 μg to about 40 μg,about 40 μg to about 50 μg, about 50 μg to about 65 μg of a vaccine).48. The microneedle of any one of the preceding claims, wherein thelength of the microneedle is between about 350 μm to about 1500 μm((e.g., about 350 μm, about 400 μm, about 450 μm, about 500 μm, about550 μm, about 600 μm, about 650 μm, about 700 μm, about 750 μm, about800 μm, about 850 μm, about 900 μm, about 950 μm, about 1000 μm, about1050 μm, about 1100 μm, about 1150 μm, about 1200 μm, about 1250 μm,about 1300 μm, about 1350 μm, about 1400 μm, about 1450 μm, about 1500μm).
 49. The microneedle of any one of the preceding claims, wherein theheight of the implantable sustained-release tip may extend toapproximately half of the full height of the microneedle.
 50. Themicroneedle of any one of the preceding claims, wherein the height ofthe implantable sustained-release tip is between about 75 μm to about475 μm (e.g., about 75, about 100 μm, about 125 μm, about 150 μm, about175 μm, about 200 μm, about 225 μm, about 250 μm, about 275 μm, about300 μm, about 325 μm, about 375 μm, about 400 μm, about 425 μm, or about475 μm).
 51. The microneedle of any one of the preceding claims, whereinthe implantable sustained-release tip comprises a tip radius betweenabout 0.5 μm to about 25 μm (e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 μm).
 52. The microneedle of any one of the precedingclaims, wherein the implantable sustained-release tip comprises a tipradius between about 5 μm to about 10 μm (e.g., about 5, 6, 7, 8, 9, or10 μm).
 53. The microneedle of any one of the preceding claims, whereinthe implantable sustained-release tip comprises an angle between about 5degrees and about 45 degrees (e.g., about 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, or 45 degrees).
 54. The microneedle of any one of thepreceding claims, wherein the backing is chosen from a solid support,e.g., a paper-based material, a plastic material, a polymeric material,or a polyester-based material (e.g., a Whatman 903 paper, a polymerictape, a plastic tape, an adhesive-backed polyester tape, or othermedical tape).
 55. The microneedle of any one of the preceding claims,wherein the implantable sustained-release tip comprises an influenzavaccine.
 56. The microneedle of claim 55, wherein the influenza vaccinecomprises an influenza A vaccine, an influenza B vaccine, an influenza Cvaccine, and/or an influenza D vaccine.
 57. The microneedle of claim 55or 56, wherein the influenza vaccine comprises an influenza A vaccine,optionally wherein the influenza A vaccine is a H1N1 vaccine and/or aH3N2 vaccine.
 58. The microneedle of any one of claims 55-57, whereinthe influenza vaccine comprises an influenza B vaccine, optionallywherein the influenza B vaccine is an B/Yamagata lineage and/or theB/Victoria lineage vaccine.
 59. The microneedle of any one of claims55-58, wherein the influenza vaccine comprises an influenza A vaccine(e.g., a H1N1 vaccine and/or a H3N2 vaccine) and an influenza B vaccine(e.g., an B/Yamagata lineage and/or the B/Victoria lineage vaccine). 60.A device, e.g., an array or patch, comprising a plurality ofmicroneedles (e.g., two or more microneedles as described herein), e.g.,a plurality of microneedles according to any one of claims 1-59.
 61. Thedevice of claim 60, wherein the microneedles of the plurality are thesame, e.g., comprise the same implantable sustained-release tip, e.g.,comprising the same therapeutic agent, e.g., the same immunogen, antigenor vaccine.
 62. The device of claim 60, wherein two or more of themicroneedles of the plurality are different, e.g., comprise two or moredifferent implantable sustained-release tips, e.g., comprising two ormore therapeutic agents, e.g., comprising a combination of two or moreimmunogens, antigens or vaccines, with or without one or more adjuvants.63. The device of claim 60, which comprises at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or more of a first implantable sustained-releasetip relative to a further (e.g., second, third, fourth, fifth)implantable sustained-release tip.
 64. The device of claim 60, wherein atotal dosage amount (e.g., a standard dose) of a vaccine, antigen,and/or immunogen is divided between the plurality of microneedles (e.g.,within a patch), such that the implantable sustained-release microneedletip can comprises at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, or 25% or more of the total dosage amount.
 65. The device of claims60-63, wherein the implantable microneedle tip comprises about 0.1 μg toabout 65 μg (e.g., about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9μg, about 1 μg, about 1 μg to about 10 μg, about 10 μg to about 20 μg,about 20 μg to about 30 μg, about 30 μg to about 40 μg, about 40 μg toabout 50 μg, about 50 μg to about 65 μg) of a vaccine, antigen, and/orimmunogen, as described herein.
 66. A method of providing immunity,e.g., broad-spectrum immunity, to a virus, e.g., an influenza virus,e.g., a drifted influenza A, B, C, and/or D strain, in a subjectcomprising contacting the skin of the subject with a microneedle of anyone of claims 1-59.
 67. A method of providing a sustained-release of avaccine, e.g., an influenza vaccine, in a subject comprising contactingthe skin of the subject with a microneedle of any one of claims 1-59.68. A method of enhancing an immune response to a virus, e.g., aninfluenza virus, e.g., a drifted influenza A, B, C, and/or D strain, ina subject comprising contacting the skin of the subject with amicroneedle of any one of claims 1-59.
 69. The method of any one ofclaims 66-68, wherein the implantable sustained-release tip isconfigured to release a vaccine into the skin of the subject over aperiod of time comprising at least about one week, e.g., about 1-2weeks, about 1-3 weeks, or about 1-4 weeks.
 70. The method of claim 69,wherein the implantable sustained-release tip is configured to release avaccine into the skin of the subject over a period of time comprising atleast about 4 days (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14days, or more, e.g., between about 4 days and about 2 weeks).
 71. Themethod of any one of claims 66-58, wherein immune response is a humoralimmune response comprises: (i) an elevated hemagglutination inhibition(HAI) antibody titer detectable in the blood of the subject, e.g.,detectable at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, and/or 25-weeks post immunization; (ii) anelevated anti-influenza IgG titer detectable in the blood of thesubject, e.g., detectable at least 1, 2, 3, 4, 5 and/or 6-months postimmunization, optionally wherein the elevated anti-influenza IgG titeris to a drifted influenza A, B, C, and/or D strain;
 72. The method ofclaim 71, wherein an elevated hemagglutination inhibition (HAI) antibodytiter is detectable in the blood of the subject for the duration of acomplete flu season post immunization, optionally wherein the elevatedHAI antibody titer is to a drifted influenza A, B, C, and/or D strain.73. The method of claim 71 or 72, wherein immune response is a cellularimmune response comprising an increase in the level of IFNγ secretingcell in the blood of the subject, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9,10, 11, and/or 12-weeks post immunization.
 74. A method of producing amicroneedle device, the method comprising: providing a mold including amold body with an array needle cavities having a predefined shape, e.g.,a pyramid-shaped and/or of conical-shaped needle cavities, formedtherein; filling tips of the needle cavities with a compositionconsisting of a silk fibroin, antigen solution; drying the filled tipsof the needle cavities to create releasable tips, and optionallyannealing the needle tips; filling the needle cavities of the mold witha dissolvable base solution; drying the dissolvable base solution tocreate base layers for the releasable tips; and applying a backing layerto the base layers to create a microneedle device.
 75. The method ofclaim 74, further comprising removing the microneedle device from themold.
 76. The method of claim 75, wherein the microneedle device isremoved by bending the mold away from the microneedle device.
 77. Themethod of claim 75, further comprising packaging microneedle devices ina container with low moisture vapor transmission rate with a desiccantto maintain between about 0% and about 50% (e.g., between about 0% and10%, between about 10% and about 20%, between about 20% and about 30%,between about 30% and about 40%, or between about 40% and 50%, e.g.,about 25%) relative humidity inside the package.
 78. The method of claim74, wherein the silk fibroin, antigen solution is dispensed into eachneedle cavity in the mold via nanoliter printing.
 79. The method ofclaim 78, wherein filling the tips of the needle cavities includesdispensing a solution, e.g., an antigen-silk formulation into eachneedle cavity.
 80. The method of claim 74, wherein drying the filledtips of the needle cavities includes a primary drying step and asecondary drying step.
 81. The method of claim 74, wherein filling theneedle cavities of the mold with a dissolvable base solution includes asolution of 40% w/v Hydrolyzed Gelatin and 10% w/v Sucrose in DIW. 82.The method of claim 81, wherein drying the dissolvable base solutionincludes subjecting the mold to a centrifuge at 3900 rpm for 2 minutesand topping off the needle cavities with 50 μL of base solution.
 83. Themethod of claim 74, further comprising an annealing step (e.g., beforefilling the base) after the filling the tips of the needle cavities. 84.The method of embodiment 74, further comprising a water annealing step(e.g., before filling the base) after the filling the tips of the needlecavities.
 85. The method of claim 74, wherein the backing layer includesone of a paper backing layer and an adhesive plastic tape.
 86. The useof a microneedle of any one of claims 1-59 in a method of providingimmunity to a virus, e.g., an influenza virus.
 87. The use of amicroneedle of any one of claims 1-59 in a method of providing asustained release of a vaccine, e.g., an influenza vaccine, in asubject.
 88. The use of a microneedle of any one of claims 1-59 in amethod of enhancing an immune response to a virus, e.g., an influenzavirus, in a subject.
 89. The microneedle of claim 1, wherein thedissolvable base comprising eight of gelatin, PEG, sucrose, CMC, PVP,PVA, hyaluronate, maltose, and methyl cellulose.
 90. The microneedle ofany one of the preceding claims, wherein the dissolvable base comprisesPEG.
 91. The microneedle of any one of the preceding claims, wherein thedissolvable base comprises between about 1% and about 70% polyethyleneglycol (PEG) (e.g., about 1%, about 2%, about 3%, about 4%, about 5%,about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, or about 70% PEG).
 92. A method forproviding broad-spectrum immunity to a virus, e.g., an influenza virus,in a subject, said method comprising administering a vaccine (e.g., ainfluenza vaccine) in an amount (e.g., a dosage) and/or over a timeperiod sufficient to result in broad-spectrum immunity to a virus, e.g.,results in an immune response (e.g., a cellular immune response and/or ahumoral immune response) to a drifted strain of the virus, in thesubject.
 93. The method of claim 92, wherein the vaccine is administeredin a composition for the controlled- or sustained-release of the vaccine(e.g., for the controlled- or sustained-release of one or more viralantigens as described herein).
 94. The method of claim 92, wherein thevaccine is administered by a device for the controlled- orsustained-release of the vaccine (e.g., for the controlled- orsustained-release of one or more viral antigens as described herein).95. The method of any of claims 92-94, wherein the vaccine isadministered into a subject, e.g., in to a tissue or cavity of thesubject chosen from skin, mucosa, organ tissue, muscle tissue or buccalcavity.
 96. The method of any of claims 92-95, wherein the vaccine isadministered in an amount (e.g., a dosage) and/or over a time periodsufficient to result in one or more of: (i) exposure in the subject toone or more antigens in the vaccine in an amount and/or period of timeto result in broad spectrum immunity, e.g., to result in an immuneresponse (e.g., a cellular immune response and/or a humoral immuneresponse) to a drifted strain of the virus, in the subject; or (ii) alevel of one or more antigens in the subject that is substantiallysteady, e.g., about 20%, 15%, 10%, 5%, or 1% to an amount, e.g., minimumamount, needed to result in an immune response (e.g., a cellular immuneresponse and/or a humoral immune response) to the one or more antigens.97. The method of any one of claims 93-96, wherein the composition ordevice for the controlled- or sustained-release of the vaccine is chosenfrom: a microneedle (e.g., a microneedle device, e.g., a microneedlepatch), an implantable device (e.g., a pump, e.g., a subcutaneous pump),an injectable formulation, a depot, a gel (e.g., a hydrogel), animplant, or a particle (e.g., a microparticle and/or a nanoparticle).98. The method of claim 97, wherein the device for the controlled- orsustained-release of the vaccine comprises a microneedle or microneedledevice, e.g., described herein.
 99. The method of claim 97, wherein thedevice for the controlled- or sustained-release of the vaccine comprisesa pump (e.g., a subcutaneous pump).
 100. The method of claim 97, whereinthe composition for the controlled- or sustained-release of the vaccinecomprises an injectable formulation (e.g., an injectable depotformulation).
 101. The method of claim 97, wherein the composition forthe controlled- or sustained-release of the vaccine comprises animplant.
 102. The method of claim 97, wherein the composition for thecontrolled- or sustained-release of the vaccine comprises a gel (e.g., ahydrogel).
 103. The method of any one of claims 97-102, wherein thecomposition or device for the controlled- or sustained-release of thevaccine comprises a particle (e.g., a microparticle and/or ananoparticle).
 104. The method of any one of claims 92-104, wherein thevaccine is administered, e.g., released by the composition or device forthe controlled- or sustained-release of the vaccine, e.g., into thesubject, in order to maintain a vaccine dosage (e.g., an antigenconcentration) for a period of time sufficient to result in broadspectrum immunity, e.g., to result in an immune response (e.g., acellular immune response and/or a humoral immune response) to a driftedstrain of the virus, in the subject (e.g., wherein the period of time isabout 1 to 21 days, e.g., about 5 to 10 days or about 5 to 7 days, e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or 21 days).
 105. The method of claim 104, wherein the compositionor device for the controlled- or sustained-release of the vaccinemaintains antigen release and/or level in the subject over a sustainedperiod of time.
 106. The method of claim 104, wherein the composition ordevice for the controlled- or sustained-release of the vaccine maintainsa continuous or non-continuous antigen release into the subject over asustained period of time.
 107. The method of any one of claims 92-106,wherein the vaccine is administered, e.g., released by the compositionor device for the controlled- or sustained-release, over a period oftime comprising at least about one week, e.g., about 1-2 weeks, about1-3 weeks, or about 1-4 weeks.
 108. The method of any one of claims92-107, wherein the vaccine is administered, e.g., released by thecomposition or device for the controlled- or sustained-release, over aperiod of time comprising at least about 4 days (e.g., about 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, or more, e.g.,between about 4 days and about 2 weeks, between about 4 days and about 1week).
 109. The method of any one of claims 92-109, wherein the vaccineis administered in a dosage comprising between about 0.1 μg and about 65μg per strain, e.g., 0.2 μg and about 50 μg per strain (e.g., about eachof 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, or 65 μg per strain).
 110. The method of any one of claims92-109, wherein at least about 1% of the dosage of the vaccine (e.g., atleast about 0.5% to about 10%, at least about 5% to about 15% at leastabout 10% to about 20% of the dosage), e.g., released by the compositionor device for the controlled- or sustained-release of the vaccine, e.g.,into the subject, is maintained over a period of time comprising atleast about 4 days (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14days, or more, e.g., between about 4 days and about 2 weeks, betweenabout 4 days and about 1 week).
 111. The method of any one of claims92-110, wherein the vaccine is administered, e.g., released by thecomposition or device for the controlled- or sustained-release, in aplurality of fractional doses of a total dose (e.g., a standard dose)over a time period, e.g., such that an immune response and/orbroad-spectrum immunity is achieved, wherein the amount of the vaccineadministered in each of the fractional doses is no more than 1/X,wherein X is any number, e.g., wherein X is 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50,60, 70, 80, 90, or 100 or more, of the total dose (e.g., a standarddose) of the vaccine.
 112. The method of any one of claims 92-110,wherein the vaccine is administered, e.g., released by the compositionor device for the controlled- or sustained-release of the vaccine, e.g.,into the skin of the subject, in a plurality of doses equivalent to apercentage of a total dose (e.g., a percentage of a standard dose) overa time period, e.g., such that broad-spectrum immunity is achieved,wherein the amount of the vaccine administered in each of the pluralityof doses is about X %, wherein X is any number, e.g., wherein X is 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 300, 400,or 500 or more, of the total dose (e.g., a standard dose) of thevaccine.
 113. The method of claim 111 or 112, wherein the vaccine isadministered such that broad-spectrum immunity is achieved, e.g., suchthat an immune response, e.g., a cellular immune and/or humoral immuneresponse to a drifted strain is achieved.
 114. The method of claim 111or 112, wherein the vaccine is administered as two, three, four, five,six, seven, eight, nine, ten or more fractional doses.
 115. The methodof any one of claims 111-114, wherein the total dose (e.g., the standarddose) of the vaccine is administered to achieve broad-spectrum immunity.116. The method of any one of claims 111-114, wherein less than thetotal dose (e.g., the standard dose) of the vaccine is administered toachieve broad-spectrum immunity.
 117. The method of any one of claims111-114, wherein more than the total dose (e.g., the standard dose) ofthe vaccine is administered to achieve broad-spectrum immunity.
 118. Themethod of any one of claims 111-114, wherein the amount of the vaccineadministered in each of the fractional doses is the same.
 119. Themethod of any one of claims 111-114, wherein the amount of the vaccineadministered in each of the fractional doses is different.
 120. Themethod of any one of claims 111-119, wherein the plurality of fractionaldoses is administered by intramuscular injection or intradermalinjection, e.g., to achieve controlled- or sustained-release of avaccine.
 121. The method of any one of claims 111-120, wherein each doseof the plurality of fractional doses is administered at least once ortwice a day, at least once every two days, at least once every threedays, at least once every four days, at least once every five days, atleast once every 6 days, at least one a week, or at least once a monthfor the duration of the time period.
 122. The method of any one ofclaims 92-121, wherein: (i) the vaccine comprises a first influenzastrain and administration of a dose of the first influenza strain to thesubject results in broad-spectrum immunity to a second influenza strain(e.g., a drifted influenza strain) not present in the implantablesustained-release tip or the vaccine; (ii) the vaccine comprises a firstinfluenza A strain and administration of a dose of the first influenza Astrain to the subject results in broad-spectrum immunity to a driftedinfluenza strain (e.g., a drifted influenza A, B, C, and/or D strain)not present in the implantable sustained-release tip or the vaccine;(iii) the vaccine comprises a first influenza B strain andadministration of a dose of the first influenza B strain to the subjectresults in broad-spectrum immunity to a drifted influenza strain (e.g.,a drifted influenza A, B, C, and/or D strain) not present in theimplantable sustained-release tip or the vaccine; (iv) the vaccinecomprises a first influenza C strain and administration of a dose of thefirst influenza C strain to the subject results in broad-spectrumimmunity to a drifted influenza strain (e.g., a drifted influenza A, B,C, and/or D strain) not present in the implantable sustained-release tipor the vaccine; and/or (v) the vaccine comprises a first influenza Dstrain and administration of a dose of the first influenza D strain tothe subject results in broad-spectrum immunity to a drifted influenzastrain (e.g., a drifted influenza A, B, C, and/or D strain) not presentin the implantable sustained-release tip or the vaccine.
 123. The methodof claim 122, wherein the first influenza A vaccine comprises: (i) anH1N1 (e.g., A/Michigan and/or A/California) vaccine; and/or (ii) an H3N2(e.g., A/Hong Kong and/or A/Switzerland) vaccine.
 124. The method ofclaim 122 or 123, wherein the drifted influenza A strain comprises: (i)an H1N1 strain (e.g., A/Michigan and/or A/California); and/or (ii) anH3N2 strain (e.g., A/Hong Kong and/or A/Switzerland).
 125. The method ofany one of claims 122-124, wherein: (i) the first influenza A vaccinecomprises an H1N1 vaccine to A/Michigan and the drifted influenza Astrain comprises A/California; and/or (ii) the first influenza A vaccinecomprises an H3N2 vaccine to A/Hong Kong and the drifted influenza Astrain is A/Switzerland.
 126. The method of claim 122, wherein the firstinfluenza B vaccine comprises: (i) a B/Yamagata lineage strain (e.g.,B/Phuket); and/or (ii) a B/Victoria lineage strain (e.g., B/Brisbane).127. The method of claim 122 or 126, wherein: (i) the drifted influenzaB strain is a B/Yamagata lineage strain (e.g., B/Phuket); and/or (ii)the drifted influenza B strain is a B/Victoria lineage strain (e.g.,B/Brisbane).
 128. The method of any one of claim 122, 126, or 127,wherein the first influenza B vaccine is to the B/Victoria lineagestrain B/Brisbane and the drifted influenza B strain is the B/Yamagatalineage strain B/Phuket.
 129. The method of any one of claims 92-128,wherein the immune response and/or broad-spectrum immunity comprises acellular and/or humoral immune response comprising: (i) an elevatedhemagglutination inhibition (HAI) antibody titer detectable in the bloodof the subject, e.g., detectable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 51, and/or 52-weeks or more post immunization, optionallywherein the elevated HAI antibody titer is to a drifted influenza A, B,C, and/or D strain; (ii) an elevated anti-influenza IgG titer detectablein the blood of the subject, e.g., detectable at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, and/or 12-months or more post immunization, optionallywherein the elevated anti-influenza IgG titer is to a drifted influenzaA, B, C, and/or D strain; and/or (iii) a level of antibody secretingplasma cells (ASC) against the virus, e.g., the influenza virus, e.g.,the drifted influenza A, B, C, and/or D strain, detectable in the bonemarrow of the subject, e.g., detectable at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 51, and/or 52-weeks or more post immunization. 130.The method of claim 129, wherein an elevated hemagglutination inhibition(HAI) antibody titer is detectable in the blood of the subject for theduration of a complete flu season post immunization, optionally whereinthe elevated HAI antibody titer is to a drifted influenza A, B, C,and/or D strain.
 131. The method of claim 129, wherein the percentseroconversion, e.g., based on the elevated HAI antibody titerdetectable in the blood of the subject, e.g., at 6-month postimmunization is greater than about 20% (e.g., 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more, e.g.,100%).
 132. The method of any one of claims 92-131, whereinbroad-spectrum immunity comprises a cellular immune response comprisingan increase in the level of IFNγ secreting cell in the blood of thesubject, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51,and/or 52-weeks or more post immunization.
 133. The method of any one ofclaims 109-132, wherein the elevated HAI antibody titer, the elevatedanti-influenza IgG titer, the level of antibody secreting plasma cells(ASC) against the virus, and/or the level of IFNγ secreting cellsdetectable in the subject is greater (e.g., 1-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold,12-fold, 13-fold, 14-fold, or 15-fold or more greater) as compared tothe administration of a single-dose or a bolus administration of thevaccine.
 134. A method for providing an immune response (e.g., acellular immune response and/or a humoral immune response) and/or abroad spectrum immunity to a virus, e.g., an influenza virus, in asubject, said method comprising administering a vaccine (e.g., ainfluenza vaccine) in an amount (e.g., a dosage) and/or over a timeperiod sufficient to elicit an immune response (e.g., a cellular immuneresponse and/or a humoral immune response) to the virus, e.g., theinfluenza virus, in the subject, wherein the vaccine is administered ina composition for the controlled- or sustained-release of the vaccine(e.g., for the controlled- or sustained-release of one or more viralantigens as described herein) over a period of time comprising about 1to about 2 weeks (e.g., about 10 days).
 135. A method or vaccine regimenof any one of the preceding claims, wherein the subject (e.g., the humansubject) is a pediatric subject.
 136. A method or vaccine regimen of anyone of the preceding claims, wherein the subject (e.g., the humansubject) is an adult subject.
 137. A method or vaccine regimen of anyone of the preceding claims, wherein the subject (e.g., the humansubject) is an elderly subject.